Methods of inhibition of protein fucosylation in vivo using fucose analogs

ABSTRACT

The invention provides methods and compositions for the inhibition of fucosylation of proteins, including antibodies, in vivo by administration of a fucose analog.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/299,894 filed on Oct. 21, 2016, which is a continuation of U.S.application Ser. No. 13/814,083 filed on Feb. 4, 2013, which is thenational stage filing under 35 U.S.C. § 371 of International ApplicationNo. PCT/US2011/046857 filed Aug. 5, 2011, which claims the benefit ofU.S. Provisional Application No. 61/371,116, filed Aug. 5, 2010, thedisclosures of each are incorporated by reference herein in theirentirety.

BACKGROUND OF THE INVENTION

L-fucose, also referred to as 6-deoxy-L-galactose, is a monosaccharidethat is a component of some N- and O-linked glycans and glycolipids inanimals. (See Becker and Lowe, Glycobiology 13:41R-51R (2003).) Fucoseis typically added as a terminal modification to glycans, includingglycans attached to blood group antigens, selectins and antibodies.Fucose can be attached to glycans via α(1,2)-, α(1,3)-, α(1,4)- andα(1,6)-linkages by specific fucosyltransferases. α(1,2)-fucose linkagesare typically associated with the H-blood group antigens. α(1,3)- andα(1,4)-fucose linkages are associated with modification of Lewis^(X)antigens. α(1,6)-fucose linkages are associated with N-linked GlcNAcmolecules, such as those on antibodies.

Fucosylation of proteins is believed to play a role in mammaliandevelopment. Mice homozygous for a targeted mutation of the FX geneexhibit pleiotropic abnormalities including a lethal phenotype. Reducedrecovery of mice from heterozygous crosses was also reported. (Becker etal., Mammalian Genome 14:130-139 (2003). Aberrant protein fucosylationhas been proposed to be associated with human disease, includingup-regulation of sialyl Lewis^(X) and sialyl Lewis^(y) in cancers. Theseglycans are ligands for E- and P-selectin molecules. In it speculatedthat increases in sialyl Lewis^(X) and sialyl Lewis^(y) glycans oncancer cells increases metastases through interaction with E- andP-selectins on endothelium. Increased fucosylated glycans have also beenobserved in patients with rheumatoid arthritis. Currently, however,there are no approved therapeutic approaches targeting proteinfucosylation levels.

SUMMARY OF THE INVENTION

The methods and compositions described herein are premised in part onthe unexpected results presented in the Examples, showing that animalsadministered a fucose analog have reduced protein fucosylation.Fucosylation of antibodies and other proteins can be modulated using thefucose analogs described herein.

In one aspect, methods and compositions for the in vivo production ofdefucosylated proteins are provided. Animals, such as mammals,administered a fucose analog (having formula I, II, III, IV, V or VI)produce proteins, such as cell surface proteins, having reducedfucosylation. The reduction in fucosylation is relative to animalsuntreated with the fucose analogs having formula I, II, III, IV, V orVI, respectively.

In a related aspect, methods and compositions for the in vivo productionof antibodies and antibody derivatives with reduced core fucosylationare provided. Animals administered a fucose analog (having formula I,II, III, IV, V or VI) produce antibodies and antibody derivatives havingreduced core fucosylation (i.e., reduced fucosylation ofN-acetylglucosamine of the complex N-glycoside-linked sugar chains boundto the Fc region through the N-acetylglucosamine of the reducingterminal of the sugar chains). The reduction in core fucosylation isrelative to animals untreated with the fucose analogs of having formulaI, II, III, IV, V or VI, respectively.

In another aspect, pharmaceutical compositions containing fucose analogsand formulated for administration to a target animal are provided. Thefucose analogs can be formulated for administration to an animal toinhibit or reduce fucosylation in vivo.

These and other aspects of the present invention may be more fullyunderstood by reference to the following detailed description,non-limiting examples of specific embodiments, and the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of administration of fucose analogs (via ipinjection) on antibody fucosylation. Dot blots are shown on the leftpanel and a graph is shown on the right panel. The dot blot proteinloading levels (upper left) and fucose-specific bioluminescence (lowerleft) for antibody cAC10 standards (lower dot blot, left most dashedrectangle and corresponding columns of upper dot blot), untreatedcontrol (lower dot blot, second dashed rectangle from the left andcorresponding column of upper dot blot), and alkynyl fucose (SGD-1887;lower dot blot, middle dashed rectangle and corresponding column ofupper dot blot), alkynyl fucose peracetate (SCD-1890; lower dot, blot,second dashed rectangle from the right and corresponding column of upperdot blot), and 2-fluorofucose (SGD-2083; lower dot blot, right mostrectangle and corresponding column of upper dot blot). After correctingfor loading level, the % fucosylation is shown on the graph at theright.

FIG. 2 shows the effects on antibody core fucosylation of administrationof fucose analogs via drinking water. The graphs show % fucosylation ofantibodies as a determined by gas chromatograph (GC): panels A and Bshow fucosylation levels of the anti-KLH antibodies (Abs) isolated fromthe treated groups while panels C and D show the fucosylation levels ofthe remaining (non-KLH-specific) IgG antibodies. Panels A and C show thepercent fucosylation of each animal determined using a purified antibodystandard curve (0-100% fucosylation). Panels B and D show thefucosylation level of the treated groups as a percentage of the averageuntreated control group value.

FIG. 3 shows the effects on antibody core fucosylation of administrationof fucose analogs via drinking water. In this figure, fucosylationlevels of the non-KLH-specific antibodies are shown. Dot blots ofprotein loading levels (upper left) and fucose specific bioluminescence(lower left) are shown for cAC10 standards (upper and lower dot blots,left most rectangle), untreated control (upper and lower dot blots,second from the left (upper) and right rectangles), and 2-fluorofucose(upper and lower dot blots, second from the left (lower) and second fromthe right rectangles (upper and lower)). After correcting for loadinglevel, the % fucosylation is shown in the graph on the right.

FIG. 4 shows the effects of different doses of 2-fluorofucose,administered via drinking water, on antibody core fucosylation. The dotblots show protein loading levels (left) and fucose specificbioluminescence (middle) for untreated control and 1, 10, and 100 mMSGD-2083 (as indicated). The % fucosylation compared to untreated isshown in the graph on the right.

FIG. 5 shows the effects of administration of 2-fluorofucose) oncirculating white blood cells and neutrophils. Panel A. Blood sampleswere collected from individual mice, and the white cell count wasdetermined by counting on a hemacytometer using Turk's solution ofexclude red blood cells. Panel B. To determine neutrophil counts, thepercentage of white blood cells that were Gr-1+ was determined by flowcytometry and applied to the total cell count determined in (A). PanelC. A pool of lymph nodes was collected from individual mice, single cellsuspensions were prepared and cells were counted on a hemacytometer.Symbols represent individual mice (n=3 per group; diamonds, untreated;squares, 1 mM 2-fluorofucose (SGD-2083); triangles, 10 mM2-fluorofucose; circles, 100 mM 2-fluorofucose).

FIG. 6 shows the effects of administration of 2-fluorofucose onE-selectin binding to neutrophils. Panel A. An example of neutrophilidentification by flow cytometry. Cells were gated on forward and sidescatter to include live white blood cells and then applied to thehistogram depicting Gr-1 staining to identify neutrophils. The positivecells were gated, the percentage positive cells determined (used forcell counts in FIG. 5B), and the gate was applied to the histograms in(B). Panel B. Examples of E-selectin binding to neutrophils from anuntreated animal (left) and an animal treated with orally administered2-fluorofucose (SGD-2083) at 100 mM (right). Grey histograms showE-selectin binding and the dotted lines show binding of the secondaryreagent alone. The geometric mean fluorescent intensity was determinedfor E-selectin binding. Panel C. Geometric mean fluorescent intensity ofE-selectin binding was determined for each animal as in (B) and comparedbetween groups (n=3, per group; error bars represent standarddeviation).

FIG. 7 shows the effects on protein fucosylation for cell lines culturedwith certain fucose analogs. The LS174T, PC-3, Ramos, HL-60cy and Caki-1cell lines were examined.

FIG. 8 shows the effects of administration of fucose analogs to mousexenograft cancer models. The results of mouse xenograft models with LS174T, PC-3, Ramos, HL-60 and Caki-1 cell lines (pre-treated with2-flurofucose (SGD-2083)), are shown in panels A-E, respectively. Theresults of a mouse xenograft model with an untreated LS174T cell linesare shown in panel F.

FIG. 9 shows the study design (panel A) and results (panel B) of a tumorvaccine model based on preimmunization with killed A20 murine lymphomacells, followed by challenge with live A20 cells with or withoutadministration of a fucose analog, (2-fluorofucose).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibody” refers to (a) immunoglobulin polypeptides andimmunologically active portions of immunoglobulin polypeptides, i.e.,polypeptides of the immunoglobulin family, or fragments thereof, thatcontain an antigen binding site(s) that immunospecifically binds to aspecific antigen and have an Fc domain comprising a complexN-glycoside-linked sugar chain(s), or (b) conservatively substitutedderivatives of such immunoglobulin polypeptides or fragments thatimmunospecifically bind to the antigen. Antibodies are generallydescribed in, for example, Harlow & Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1988).

An “antibody derivative” means an antibody, as defined above (includingan antibody fragment), or Fc domain or region of an antibody comprisinga complex N-glycoside linked sugar chain, that is modified by covalentattachment of a heterologous molecule such as, e.g., by attachment of aheterologous polypeptide (e.g., a ligand binding domain of heterologousprotein), or by glycosylation (other than core fucosylation),deglycosylation (other than non-core fucosylation), acetylation,phosphorylation or other modification not normally associated with theantibody or Fc domain or region.

The term “monoclonal antibody” refers to an antibody that is derivedfrom a single cell clone, including any eukaryotic or prokaryotic cellclone, or a phage clone, and not the method by which it is produced.Thus, the term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology.

The term “Fc region” refers to the constant region of an antibody, e.g.,a C_(H)1-hinge-C_(H)2-C_(H)3 domain, optionally having a C_(H)4 domain,or a conservatively substituted derivative of such an Fc region.

The term “Fc domain” refers to the constant region domain of anantibody, e.g., a C_(H)1, hinge, C_(H)2, C_(H)3 or C_(H)4 domain, or aconservatively substituted derivative of such an Fc domain.

An “antigen” is a molecule to which an antibody or antibody derivativespecifically binds.

The terms “specific binding” and “specifically binds” mean that theantibody or antibody derivative will bind, in a highly selective manner,with its corresponding target antigen and not with the multitude ofother antigens. Typically, the antibody or antibody derivative bindswith an affinity of at least about 1×10⁻⁷ M, and preferably 10⁻⁸ M to10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M and binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.

The terms “inhibit” or “inhibition of” means to reduce by a measurableamount, or to prevent entirely.

As used herein, “alkynyl fucose peracetate” refers to any or all formsof alkynyl fucose (5-ethynylarabinose) with acetate groups on positionsR¹⁻⁴ (see formula I and II, infra), including6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate, includingthe (2S,3S,4R,5R,6S) and (2R,3S,4R,5R,6S) isomers, and5-((S)-1-hydroxyprop-2-ynyl)-tetrahydrofuran-2,3,4-triyl tetraacetate,including the (2S,3S,4R,5R) and (2R,3S,4R,5R) isomers, and the aldoseform, unless otherwise indicated by context. The terms “alkynyl fucosetriacetate”, “alkynyl fucose diacetate” and “alkynyl fucose monoacetate”refer to the indicated tri-, di- and mono-acetate forms of alkynylfucose, respectively.

Unless otherwise indicated by context, the term “alkyl” refers to anunsubstituted saturated straight or branched hydrocarbon having from 1to 20 carbon atoms (and all combinations and subcombinations of rangesand specific numbers of carbon atoms therein), unless otherwisespecified. An alkyl group of 1 to 3, 1 to 8 or 1 to 10 carbon atoms ispreferred. Examples of alkyl groups are methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl- 1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

Alkyl groups, whether alone or as part of another group, whensubstituted can be substituted with one or more groups, preferably 1 to3 groups (and any additional substituents selected from halogen),including, but not limited to: halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, ═O, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, or aryl.

Unless otherwise indicated by context, the terms “alkenyl” and “alkynyl”refer to unsubstituted or optionally substituted (were indicated)straight and branched carbon chains having from 2 to 20 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from 2 to 3, 2 to 4, 2 to 8 or 2 to 10carbon atoms being preferred. An alkenyl chain has at least one doublebond in the chain and an alkynyl chain has at least one triple bond inthe chain. Examples of alkenyl groups include, but are not limited to,ethylene or vinyl, allyl, -1 butenyl, -2 butenyl, -isobutylenyl, -1pentenyl, -2 pentenyl, 3-methyl-1-butenyl, -2 methyl 2 butenyl, and -2,3dimethyl 2 butenyl. Examples of alkynyl groups include, but are notlimited to, acetylenic, propargyl, acetylenyl, propynyl, -1 butynyl, -2butynyl, -1 pentynyl, -2 pentynyl, and -3 methyl 1 butynyl.

Alkenyl and alkynyl groups, whether alone or as part of another group,when substituted can be substituted with one or more groups, preferably1 to 3 groups (and any additional substituents selected from halogen),including but not limited to: halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, ═O, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl, —C₂-C alkenyl, —C₂-C₈alkynyl, or aryl.

Unless otherwise indicated by context, the term “alkylene” refers to anunsubstituted saturated branched or straight chain hydrocarbon radicalhaving from 1 to 20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein),with from 1 to 8 or 1 to 10 carbon atoms being preferred and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkane. Typicalalkylenes include, but are not limited to, methylene, ethylene,propylene, butylene, pentylene, hexylene, heptylene, ocytylene,nonylene, decalene, 1,4-cyclohexylene, and the like.

Alkylene groups, whether alone or as part of another group, whensubstituted can be substituted with one or more groups, preferably 1 to3 groups (and any additional substituents selected from halogen),including, but not limited to: halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,═O, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ is independentlyselected from H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of an alkenyl group (as described above), andhaving two monovalent radical centers derived by the removal of twohydrogen atoms from the same or two different carbon atoms of a parentalkene. An “alkenylene” group can be unsubstituted or optionallysubstituted (were indicated), as described above for alkenyl groups, Insome embodiments, an “alkenylene” group is not substituted.

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of an alkynyl group (as described above), andhaving two monovalent radical centers derived by the removal of twohydrogen atoms from the same or two different carbon atoms of a parentalkyne. An “alkynylene” group can be unsubstituted or optionallysubstituted (were indicated), as described above for alkynyl groups, Insome embodiments, an “alkynylene,” group is not substituted.

Unless otherwise indicated by context, the term “aryl” refers to asubstituted or unsubstituted monovalent aromatic hydrocarbon radical of6-20 carbon atoms (and all combinations and subcombinations of rangesand specific numbers of carbon atoms therein) derived by the removal ofone hydrogen atom from a single carbon atom of a parent aromatic ringsystem. Some aryl groups are represented in the exemplary structures as“Ar”. Typical aryl groups include, but are not limited to, radicalsderived from benzene, substituted benzene, phenyl, naphthalene,anthracene, biphenyl, and the like.

An aryl group, whether alone or as part of another group, can beoptionally substituted with one or more, preferably 1 to 5, or even 1 to2 groups including, but not limited to: halogen, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkenyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, —NO₂,—NH2, —NH(R′), —N(R′)₂ and —CN; where each R′ is independently selectedfrom H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

Unless otherwise indicated by context, the term “heterocycle” refers toa substituted or unsubstituted monocyclic ring system having from 3 to7, or 3 to 10, ring atoms (also referred to as ring members) wherein atleast one ring atom is a heteroatom selected from N, O, P, or S (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms and heteroatoms therein). The heterocycle can have from 1to 4 ring heteroatoms independently selected from N, O, P, or S. One ormore N, C, or S atoms in a heterocycle can be oxidized. A monocyclicheterocycle preferably has 3 to 7 ring members (e.g., 2 to 6 carbonatoms and 1 to 3 heteroatoms independently selected from N, O, P, or S).The ring that includes the heteroatom can be aromatic or non-aromatic.Unless otherwise noted, the heterocycle is attached to its pendant groupat any heteroatom or carbon atom that results in a stable structure.

Heterocycles are described in Paquette, “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.8:2:5566 (1960). Examples of “heterocycle,” groups include by way ofexample and not limitation pyridyl, dihydropyridyl, tetrahydropyridyl(piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,pyrazolyl, imidazolyl, tetrazolyl, fucosyl, azirdinyl, azetidinyl,oxiranyl, oxetanyl, and tetrahydrofuranyl.

A heterocycle group, whether alone or as part of another group, whensubstituted can be substituted with one or more groups, preferably 1 to2 groups, including but not limited to: —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C8alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, —NH₂,—NH(R′), —N(R′)₂ and —CN; where each R′ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

By way of example and not limitation, carbon-bonded heterocycles can bebonded at the following positions: position 2, 3, 4, 5, or 6 of apyridine; position 3, 4, 5, or 6 of a pyridazine; position 2, 4, 5, or 6of a pyrimidine; position 2, 3, 5, or 6 of a pyrazine; position 2, 3, 4,or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole ortetrahydropyrrole; position 2, 4, or 5 of an oxazole, imidazole orthiazole; position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole;position 2 or 3 of an aziridine; or position 2, 3, or 4 of an azetidine.Exemplary carbon bonded heterocycles can include 2-pyridyl, 3-pyridyl,4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl,6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole;position 2 of a isoindole, or isoindoline; and position 4 of amorpholine. Still more typically, nitrogen bonded heterocycles include1-aziridyl, 1-azetidyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and1-piperidinyl.

Unless otherwise noted, the term “carbocycle,” refers to a substitutedor unsubstituted, saturated or unsaturated non-aromatic monocyclic ringsystem having from 3 to 6 ring atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein)wherein all of the ring atoms are carbon atoms.

Carbocycle groups, whether alone or as part of another group, whensubstituted can be substituted with, for example, one or more groups,preferably 1 or 2 groups (and any additional substituents selected fromhalogen), including, but not limited to: halogen, C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —NH₂,—NH(R′), —N(R′)₂ and —CN; where each R′ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

Examples of monocyclic carbocylic substituents include cyclopropyl,cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl,1-cyclohex-3-enyl, cycloheptyl, cyclooctyl, -1,3-cyclohexadienyl,-1,4-cyclohexadienyl, -1,3 -cycloheptadienyl, -1,3,5-cycloheptatrienyl,and -cyclooctadienyl.

When any variable occurs more than one time in any constituent or in anyformula, its definition in each occurrence is independent of itsdefinition at every other. Combinations of substituents and/or variablesare permissible only if such combinations result in stable compounds.

Unless otherwise indicated by context, a hyphen (-) designates the pointof attachment to the pendant molecule. Accordingly, the term “—(C₁-C₁₀alkylene)aryl” or “—C₁C₁₀ alkylene(aryl)” refers to a C₁-C₁₀ alkyleneradical as defined herein wherein the alkylene radical is attached tothe pendant molecule at any of the carbon atoms of the alkylene radicaland one of the hydrogen atom bonded to a carbon atom of the alkyleneradical is replaced with an aryl radical as defined herein.

When a particular group is “substituted”, that group may have one ormore substituents, preferably from one to five substituents, morepreferably from one to three substituents, most preferably from one totwo substituents, independently selected from the list of substituents.The group can, however, generally have any number of substituentsselected from halogen.

It is intended that the definition of any substituent or variable at aparticular location in a molecule be independent of its definitionselsewhere in that molecule. It is understood that substituents andsubstitution patterns on the compounds of this invention can be selectedby one of ordinary skill in the art to provide compounds that are activeand chemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “pharmaceuticallycompatible ingredient” refers to a pharmaceutically acceptable diluent,adjuvant, excipient, or vehicle with which the fucose analog isadministered.

“Small electron-withdrawing groups” refers to any substituent that hasgreater electronegativity at the site of substituent attachment than,e.g., a hydrogen atom or hydroxy group or relative to the substituentpresent in fucose at that site. Generally, the smallelectron-withdrawing group has 10 or fewer atoms (other than hydrogen)and includes groups such as nitro; cyano and cyanoalkyl (e.g.,—CH₂CH₂CN); halogens; acetylene or other alkynes or halo alkynes (e.g.,—C≡CCF₃); alkenes or halo alkenes; allenes; carboxylic acids, ester,amides and halo substituted forms thereof; sulfonic and phosphonicacids, esters and amides, and halo substituted forms thereof; haloalkylgroups (e.g., —CF₃, —CHF₂, —CH₂CF₃), acyl and haloacyl groups (e.g.,—C(O)CH₃ and —C(O)CF₃); alkylsulfonyl and haloalkylsulfonyl (e.g.,—S(O)₂alkyl and —S(O)₂haloalkyl); aryloxy (e.g., phenoxy and substitutedphenoxy); aralkyloxy (e.g, benzyloxy and substituted benzyloxy); andoxiranes. Preferred small electron-withdrawing groups are those having8, 7 or 6 or fewer atoms (other than hydrogen).

The fucose analogs are typically substantially pure from undesiredcontaminant. This means that the analog is typically at least about 50%w/w (weight/weight) purity, as well as being substantially free frominterfering proteins and other contaminants. Sometimes the agents are atleast about 80% w/w and, more preferably at least 90% or about 95% w/wpurity. Using conventional purification techniques, homogeneous productof at least 99% w/w can be obtained.

General

The invention provides methods and compositions for reducing proteinfucosylation in an animal. The methods are premised in part on theunexpected results presented in the Examples showing that administeringa fucose analog to a subject (e.g., a mammal) results in an antibody orantibody derivative having reduced core fucosylation, and other proteinsalso having reduced fucosylation. “Reduced fucosylation” in the contextof proteins generally refers to reduced addition of fucose to glycansvia α(1,2)-, α(1,3)-, α(1,4)- and α(1,6)-linkages. “Core fucosylation”in the context of an antibody refers to addition of fucose(“fucosylation”) to N-acetylglucosamine (“GlcNAc”) at the reducingterminal of an N-linked glycan of an antibody. “Reduced corefucosylation” in the context of an antibody refers to a reduction offucose linked to N-acetylglucosamine (“GlcNAc”) at the reducing terminalof an N-linked glycan of an antibody, as compared to an untreatedanimal.

In the various aspects described herein, the animal to which the fucoseanalog is administered is typically a mammal and is preferably human.The invention therefore further provides methods and compositions forreducing protein fucosylation in a mammal, such as a human.

In other aspects, pharmaceutical compositions of fucose analogs andpharmaceutical excipients are provided in which an effective amount of afucose analogs) is in admixture with the excipients, suitable foradministration to a animal. In some embodiments, the fucose analog is indry form (e.g., lyophilized), optionally with stabilizers that enhancethe composition stability for longer term storage. In some embodiments,a pharmaceutical composition of a fucose analogs and pharmaceuticalexcipients is formulated for administration to a mammal. In some furtherembodiments, a pharmaceutical composition of a fucose analogs andpharmaceutical excipients is formulated for administration to a human.

In some embodiments, fucosylation of complex N-glycoside-linked sugarchains bound to the Fc region (or domain) of an antibody is reduced. Asused herein, a “complex N-glycoside-linked sugar chain” is typicallybound to asparagine 297 (according to the numbering system of Kabat),although a complex N-glycoside linked sugar chain can also be linked toother asparagine residues. As used herein, the complexN-glycoside-linked sugar chain has a bianntennary composite sugar chain,mainly having the following structure:

where ± indicates the sugar molecule can be present or absent, and thenumbers indicate the position of linkages between the sugar molecules.In the above structure, the sugar chain terminal which binds toasparagine is called a reducing terminal (at right), and the oppositeside is called a non-reducing terminal. Fucose is usually bound toN-acetylglucosamine (“GlcNAc”) of the reducing terminal, typically by anα1,6 bond (the 6-position of GlcNAc is linked to the 1-position offucose). “Gal” refers to galactose, and “Man” refers to mannose.

A “complex N-glycoside-linked sugar chain” excludes a high mannose typeof sugar chain, in which only mannose is incorporated at thenon-reducing terminal of the core structure, but includes 1) a complextype, in which the non-reducing terminal side of the core structure hasone or more branches of galactose-N-acetylglucosamine (also referred toas “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAcoptionally has a sialic acid, bisecting N-acetylglucosamine or the like;or 2) a hybrid type, in which the non-reducing terminal side of the corestructure has both branches of the high mannose N-glycoside-linked sugarchain and complex N-glycoside-linked sugar chain.

In some embodiments, the “complex N-glycoside-linked sugar chain”includes a complex type in which the non-reducing terminal side of thecore structure has zero, one or more branches ofgalactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and thenon-reducing terminal side of Gal-GlcNAc optionally further has astructure such as a sialic acid, bisecting N-acetylglucosamine or thelike, but excludes chains with a high mannose component.

According to the present methods, typically only a minor amount offucose is incorporated into the sugar chain(s) (e.g., a glycan orcomplex N-glycoside-linked sugar chains) after administering a fucoseanalog. For example, in various embodiments, less than about 60%, lessthan about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,or less than about 1% of the antibodies in the serum of the animal(e.g., a mammal, such as a human) are core fucosylated, as compared toan animal not receiving the fucose analog. In some embodiments,substantially none (i.e., less than 0.5%) of the antibodies in the serumof the animal are not core fucosylated, as compared to an animal notreceiving the fucose analog.

In some embodiments, protein fucosylation is reduced by about 60%, byabout 50%, by about 40%, by about 30%, by about 20%, by about 15%, byabout 10%, by about 5%, or by about 1% for cell surface proteins in theanimal (e.g., a mammal, such as a human) are fucosylated, as compared toan animal not receiving the fucose analog. In some embodiments, proteinfucosylation via α(1,2)-linkage is reduced by about 60%, by about 50%,by about 40%, by about 30%, by about 20%, by about 15%, by about 10%, byabout 5%, or by about 1% for cell surface proteins in the animal (e.g.,a mammal, such as a human) are fucosylated, as compared to an animal notreceiving the fucose analog.

In some embodiments, protein fucosylation via α(1,3)-linkage is reducedby about 60%, by about 50%, by about 40%, by about 30%, by about 20%, byabout 15%, by about 10%. by about 5%, or by about 1% for cell surfaceproteins in the animal (e.g., a mammal, such as a human) arefucosylated, as compared to an animal not receiving the fucose analog.In some embodiments, protein fucosylation via α(1,4)-linkage is reducedby about 60%, by about 50%, by about 40%, by about 30%, by about 20%, byabout 15%, by about 10%, by about 5%, or by about 1% for cell surfaceproteins in the animal (e.g., a mammal, such as a human) arefucosylated, as compared to an animal not receiving the fucose analog.

In some embodiments, protein fucosylation via α(1,6)-linkage is reducedby about 60%, by about 50%, by about 40%, by about 30%, by about 20%, byabout 15%, by about 10%, by about 5%, or by about 1% for cell surfaceproteins in the animal (e.g., a mammal, such as a human) arefucosylated, as compared to an animal not receiving the fucose analog.

In some embodiments, fucosylation of white blood cells in the serum ofthe animal (e.g., a mammal, such as a human) is reduced by at leastabout 60%, at least about 50%, at least about 40%, at least about 30%,at least about 20%, at least about 15%, at least about 10%, or at leastabout 5%, as compared to an animal not receiving the fucose analog. Insome embodiments, fucosylation via α(1,3) linkages of white blood cellsin the serum of the animal (e.g., a mammal, such as a human) is reducedby at least about 60%, at least about 50%, at least about 40%, at leastabout 30%, at least about 20%, at least about 15%, at least about 10%,or at least about 5%, as compared to an animal not receiving the fucoseanalog. In some embodiments, fucosylation via α(1,4) linkages of whiteblood cells in the serum of the animal (e.g., a mammal, such as a human)is reduced by at least about 60%, at least about 50%, at least about40%, at least about 30%, at least about 20%, at least about 15%, atleast about 10%, or at least about 5%, as compared to an animal notreceiving the fucose analog.

In some embodiments, fucosylation of antibodies in the serum of theanimal (e.g., a mammal, such as a human) is reduced by at least about60%, at least about 50%, at least about 40%, at least about 30%, atleast about 20%, at least about 15%, at least about 10%, or at leastabout 5%, as compared to an animal not receiving the fucose analog.

In certain embodiments, only a minor amount of a fucose analog (or ametabolite or product of the fucose analog) is incorporated into glycans(e.g., the complex N-glycoside-linked sugar chain(s)) of the antibody,antibody derivative or other glycans of proteins. For example, invarious embodiments, less than about 60%, less than about 40%, less thanabout 30%, less than about 20%, less than about 15%, less than about10%, less than about 5%, or less than about 1% of the fucose analog (ora metabolite or product of the fucose analog) is incorporated intoglycans of the antibodies in the serum of the animal, as compared to ananimal not receiving the fucose analog. in some embodiments, less thanabout 60%, less than about 40%, less than about 30%, less than about20%, less than about 15%, less than about 10%, less than about 5%, orless than about 1% of the fucose analog (or a metabolite or product ofthe fucose analog) is incorporated into glycans of cell surface proteinsof the animal, as compared to an animal not receiving the fucose analog.

In some embodiments, less than about 60%, less than about 40%, less thanabout 30%, less than about 20%, less than about 15%, less than about10%, less than about 5%, or less than about 1% of the fucose analog (ora metabolite or product of the fucose analog) is incorporated intoglycans of white blood cells in the serum of the animal, as compared toan animal not receiving the fucose analog.

Fucose Analogs

Suitable fucose analogs for the methods of the present invention(identified below as Formula I, II, III, IV, V and VI) are those thatcan be safely administered to a

mammal in an amount effective to inhibit core fucosylation of complexN-glycoside-linked sugar chains of antibodies or antibody derivatives.Fucose analogs are described in Published US Patent Application2009-0317869 that reduce the incorporation of fucose into complexN-glycoside-linked sugar chains of antibodies or antibody derivativesproduced by host cells in vitro. The fucose analog can be given to asubject animal (e.g., a mammal) by parental, orally or other suitablemode of administration.

In some embodiments, a fucose analog (or an intracellular metabolite orproduct of the fucose analog) inhibits an enzyme(s) in the fucosesalvage pathway. (As used herein, an intracellular metabolite can be,for example, a GDP-modified analog or a fully or partially de-esterifiedanalog. A product can be, for example, a fully or partiallyde-esterified analog.) For example, a fucose analog (or an intracellularmetabolite or product of the fucose analog) can inhibit the activity offucokinase, or GDP-fucose-pyrophosphorylase. In some embodiments, afucose analog (or an intracellular metabolite or product of the fucoseanalog) inhibits fucosyltransferase (such as a 1,2-fucosyltransferase,1,3-fucosyltransferase, 1,4-fucosyltransferase, or1,6-fucosyltransferase (e.g., the FUT8 protein)). In some embodiments, afucose analog (or an intracellular metabolite or product of the fucoseanalog) can inhibit the activity of an enzyme in the de novo syntheticpathway for fucose. For example, a fucose analog (or an intracellularmetabolite or product of the fucose analog) can inhibit the activity ofGDP-mannose 4,6-dehydratase or/or GDP-fucose synthetase. In someembodiments, the fucose analog (or an intracellular metabolite orproduct of the fucose analog) can inhibit fucose transporter (e.g.,GDP-fucose transporter).

In some embodiments, the fucose analog has the following formula (I) or(II):

or a biologically acceptable salt or solvate of the analog, wherein eachof formula (I) or (II) can be the alpha or beta anomer or thecorresponding aldose form. In the above formulae, each of R¹-R⁴ isindependently selected from the group consisting of —OH, —OC(O)H,—OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀ alkynyl,—OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl), —OC(O)C₂-C₁₀alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl), —OC(O)C₁-C₁₀alkylene(heterocycle), —OC(O)C₂-C₁₀ alkenylene(heterocycle),—OC(O)C₂-C₁₀ alkynylene(heterocycle), —OC(O)CH₂O(CH₂CH₂O)_(n)CH₃,—OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃, —O-tri-C₁-C₃ alkyl silyl, —OC₁-C₁₀ alkyl,—OCH₂OC(O) alkyl, —OCH₂OC(O) alkenyl, —OCH₂OC(O) alkynyl, —OCH₂OC(O)aryl, —OCH₂OC(O) heterocycle, —OCH₂OC(O)O alkyl, —OCH₂OC(O)O alkenyl,—OCH₂OC(O)O alkynyl, —OCH₂OC(O)O aryl and —OCH₂OC(O)O heterocycle,wherein each n is an integer independently selected from 0-5; and R⁵ isselected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH,—C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is F, Br, Cl orI), —CHX₂ (wherein each X is F, Br or CI) and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), wherein:each of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)aryl, —OC(O)heterocycle,—OC(O)C₁-C₁₀ alkylene(aryl), —OC(O)C₁-C₁₀ alkylene(heterocycle),—OC(O)CH₂O(CH₂CH₂O)_(n)CH₃, —OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃, —O-tri-C₁-C₃silyl, —OC₁-C₁₀ alkyl, —OCH₂OC(O) alkyl, —OCH₂OC(O)O alkyl, —OCH₂OC(O)aryl, and —OCH₂OC(O)O aryl, wherein each n is an integer independentlyselected from 0-5; and R⁵ is selected from the group consisting of—C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X(wherein X is F, Br, Cl or I), —CHX₂ (wherein each X is F, Br or Cl),and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl),—OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl),—OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), and —OC(O)C₂-C₁₀ alkynylene(heterocycle); andR⁵ is selected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH,—C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is F, Br, Cl orI), —CHX₂ (wherein each X is F, Br or Cl), and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—O-tri-C₁-C₃ silyl and —OC₁-C₁₀ alkyl; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN,—CH₂CN, —CH₂X (wherein X is Br, Cl or I), and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OCH₂OC(O) alkyl, —OCH₂OC(O) alkenyl, —OCH₂OC(O) alkynyl, —OCH₂OC(O)aryl, —OCH₂OC(O) heterocycle, —OCH₂OC(O)O alkyl, —OCH₂OC(O)O alkenyl,—OCH₂OC(O)O alkynyl, —OCH₂OC(O)O aryl, and —OCH₂OC(O)O heterocycle; andR⁵ is selected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH,—C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is F, Br, Cl orI), —CHX₂ (wherein each X is F, Br or Cl), and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl),—OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl),—OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), and —OC(O)C₂-C₁₀ alkynylene(heterocycle); andR⁵ is selected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH,—C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl),—OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl),—OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), and —OC(O)C₂-C₁₀ alkynylene(heterocycle); andR⁵ is selected from the group consisting of —CH₂F, —CH₂I, —CH₂Br, and—CH₂Cl.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl),—OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl),—OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), and —OC(O)C₂-C₁₀ alkynylene(heterocycle); andR⁵ is selected from the group consisting of —CHF₂, —CHBr₂, and —CHCl₂.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl),—OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl),—OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), and —OC(O)C₂-C₁₀ alkynylene(heterocycle); andR⁵ is selected from the group consisting of —C≡CH, —C≡CCH₃ and —CH₂C≡CH.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl),—OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl),—OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), and —OC(O)C₂-C₁₀ alkynylene(heterocycle); andR⁵ is selected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂CN and—CO(O)CH₃.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀alkylene(aryl),—OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl),—OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), and —OC(O)C₂-C₁₀ alkynylene(heterocycle); andR⁵ is selected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂CN and—CO(O)CH₃.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R¹ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl),—OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl),—OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), and —OC(O)C₂-C₁₀ alkynylene(heterocycle); andR⁵ is selected from the group consisting of —C≡CH, —C≡CCH₃, 13CH(OAc)CH₃, —CH₂CN, and —CO(O)CH₃.

In some embodiments, the fucose analog has formula (I) or (II), whereinR⁵ is as defined herein, and each of R¹-R⁴ is hydroxyl or —OC(O)C₁-C₁₀alkyl.

In some embodiments, the fucose analog has formula (I) or (II), whereinR⁵ is as defined herein, and each of R¹-R⁴ is hydroxyl or —OAc.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OC(O)C₁-C₁₀ alkyl; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃, —CH(OAc)CH₃, —CH₂CN, —CO(O)CH₃, —CH₂F and—CHF₂

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OAc; and R⁵ is selected from the group consisting of —C≡CH,—C≡CCH₃, —CH(OAc)CH₃, —CH₂CN, —CO(O)CH₃, —CH₂F and —CHF₂

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OC(O)C₁-C₁₀ alkyl; and R⁵ is selected from the groupconsisting of —C≡CH, —CH₂F and —CHF₂.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OAc; and R⁵ is selected from the group consisting of —C≡CH,—CH₂F and —CHF₂.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OC(O)C₁-C₁₀ alkyl; and R⁵ is —C≡CH.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OAc; and R⁵ is —C≡CH.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OC(O)C₁-C₁₀ alkyl; and R⁵ is —CHF₂.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OAc; and R⁵ is —CHF₂.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is —OH or an ester selected from the group consisting of—OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀ alkynyl,—OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene(aryl), —OC(O)C₂-C₁₀alkenylene(aryl), —OC(O)C₂-C₁₀ alkynylene(aryl), —OC(O)C₁-C₁₀alkylene(heterocycle), —OC(O)C₂-C₁₀ alkenylene(heterocycle),—OC(O)C₂-C₁₀ alkynylene(heterocycle), —OC(O)CH₂O(CH₂CH₂O)_(n)CH₃ (wheren is 0-5), and —OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃ (where n is 0-5); and R⁵ isselected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH,—C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is F, Br, Cl orI), —CHX₂ (wherein each X is F, Br or CI), and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OC(O)C₁-C₁₀ alkyl; and R⁵ is —CH₂X (wherein X is F, Br, Cl orI).

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OAc; and R⁵ is —CH₂X (wherein X is F, Br, Cl or I).

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OC(O)C₁-C₁₀ alkyl; and R⁵ is —CH₂Br.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of —OHand —OAc; and R⁵ is —CH₂Br.

In some embodiments, the fucose analog has a molecular weight of lessthan 2000 daltons. In some embodiments, the fucose analog has amolecular weight of less than 1000 daltons.

In some embodiments, R⁵ is not substituted.

In some embodiments, each of R¹-R⁴ is not substituted.

In some embodiments, R⁵ is not a ketone (—C(O)alkyl).

In some embodiments, R⁵ is not —CH(CH₃)OAc.

In some embodiments, R⁵ is not —CH(CH₃)OAc, when each of R¹-R⁴ is —OAc.

In some embodiments, R⁵ is not —C≡CH.

In some embodiments, R⁵ is not —C≡CH, when any of R¹-R⁴ is —OAc.

In some embodiments, R⁵ is not —C≡CH, when any of R¹-R⁴ is —OC(O)alkyl.

In some embodiments, R⁵ is not —C≡CH, when each of R¹-R⁴ is —OC(O)alkyl.

In some embodiments, R⁵ is not —C≡CH₃ when each of R¹-R⁴ is OH.

In some embodiments, the fucose analog is alkynyl fucose peracetate. Insome embodiments, the fucose analog is alkynyl fucose triacetate. Insome embodiments, the fucose analog is alkynyl fucose diacetate. In someembodiments, the fucose analog is mixture of alkynyl fucose peracetate,alkynyl fucose triacetate and alkynyl fucose diacetate.

In some embodiments, the fucose analog is mixture of alkynyl fucoseperacetate, alkynyl fucose triacetate, alkynyl fucose diacetate andalkynyl fucose monoacetate.

In any of the various embodiments, the fucose analog is not fucose. Insome embodiments, the fucose analog is not alkynyl fucose peracetate. Insome embodiments, the fucose analog is not galactose or L-galactose.

In another group of embodiments, the fucose analog has the followingformula (III) or (IV):

or a biologically acceptable salt or solvate thereof, wherein each offormula (III) or (IV) can be the alpha or beta anomer or thecorresponding aldose form; and wherein,

-   each of R¹-R⁴ is independently selected from the group consisting of    fluoro, chloro, —OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀    alkenyl, —OC(O)C₂-C₁₀ alkynyl, —OC(O)aryl, —OC(O)heterocycle,    —OC(O)C₁-C₁₀ alkylene(aryl), —OC(O)C₂-C₁₀ alkenylene(aryl),    —OC(O)C₂-C₁₀ alkynyl(aryl), —OC(O)C₁-C₁₀ alkylene(heterocycle),    —OC(O)C₂-C₁₀ alkenylene(heterocycle), —OC(O)C₂-C₁₀    alkynylene(heterocycle), —OCH₂OC(O) alkyl, —OCH₂OC(O)O alkyl    —OCH₂OC(O) aryl, —OCH₂OC(O)O aryl,-    —OC(O)CH₂O(CH₂CH₂O)_(n)CH₃, —OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃,    —O-tri-C₁-C₃ alkylsilyl and —OC₁-C₁₀ alkyl, wherein each n is an    integer independently selected from 0-5; and    each of R^(2a) and R^(3a) is independently selected from the group    consisting of H, F and Cl;-   R⁵ is selected from the group consisting of —CH₃, —CHF₂, —CH═C═CH₂,    —C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X    (wherein X is F, Br, Cl or I), and methoxiran;    wherein when R⁵ is other than —CH═C═CH₂, —CH₂F or —CHF₂, at least    one of R¹, R², R³, R^(2a) and R^(3a) is fluoro or chloro.

In some embodiments of formulae (III) or (IV), R¹ is F.

In some embodiments of formulae (III) or (IV), R² is F.

In some embodiments of formulae (III) or (IV), R³ is F.

In some embodiments of formulae (III) or (IV), R¹ and R² are each F.

In some embodiments of formulae (III) or (IV), R² and R^(2a) are each F.

In some embodiments of formulae (III) or (IV), R¹, R³ and R⁴ are eachindependently selected from —OH and —OC(O)C₁-C₁₀ alkyl; R² is F; and R⁵is —CH₃.

In some embodiments of formulae (III) or (IV), R¹, R³ and R⁴ are eachindependently selected from —OH and —OAc; R² is F; and R⁵ is —CH₃.

In some embodiments of formulae (III) or (IV), R¹, R³ and R⁴ are eachindependently selected from —OH and —OC(O)C₁-C₁₀ alkyl; R² is F; R^(2a)and R^(3a) are each H; and R⁵ is —CH₃.

In some embodiments of formulae (III) or (IV), R¹, R³ and R⁴ are eachindependently selected from —OH and —OAc; R² is F; R^(2a) and R^(3a) areeach H; and R⁵ is —CH₃.

In some embodiments of formulae (III) or (IV), R¹, R², R³ and R⁴ areeach independently selected from —OH and —OC(O)C₁-C₁₀ alkyl; R^(2a) andR^(3a) are each H; and R⁵ is —CHF₂.

In some embodiments of formulae (III) or (IV), R¹, R², R³ and R⁴ areeach independently selected from —OH and —OAc; R^(2a) and R^(3a) areeach H; and R⁵ is —CHF₂.

In some embodiments of formulae (III) or (IV), R¹, R², R³ and R⁴ areeach independently selected from —OH and —OC(O)C₁-C₁₀ alkyl; R^(2a) andR³¹ are each H; and R⁵ is —CH₂F.

In some embodiments of formulae (III) or (IV), R¹, R², R³ and R⁴ areeach independently selected from —OH and —OAc; R^(2a) and R^(3a) areeach H; and R⁵ is —CH₂F.

In another group of embodiments, the fucose analog has the followingformula (V) or (VI):

or a biologically acceptable salt or solvate thereof, wherein each offormula (V) or (VI) can be the alpha or beta anomer or the correspondingaldose form; and wherein,each of R¹, R², R^(2a), R³, R^(3a) and R⁴ is independently selected fromthe group consisting of —OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀alkenyl, —OC(O)C₂-C₁₀ alkynyl, —OC(O)aryl, —OC(O)heterocycle,—OC(O)C₁-C₁₀ alkylene(aryl), —OC(O)C₂-C₁₀ alkenylene(aryl), —OC(O)C₂-C₁₀alkynylene(aryl), —OC(O)C₁-C₁₀ alkylene(heterocycle), —OC(O)C₂-C₁₀alkenylene(heterocycle), —OC(O)C₂-C₁₀ alkynylene(heterocycle),—OCH₂OC(O) alkyl, —OCH₂OC(O)O alkyl, —OCH₂OC(O) aryl, —OCH₂OC(O)O aryl,—OC(O)CH₂O(CH₂CH₂O)_(n)CH₃, —OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃, —O-tri-C₁-C₃alkylsilyl, —OC₁-C₁₀ alkyl, and a small electron withdrawing group,wherein each n is an integer independently selected from 0-5;R⁵ is a member selected from the group consisting of —CH₃, —CHX₂, —CH₂X,—CH(X′)—C₁-C₄ alkyl unsubstituted or substituted with halogen,—CH(X′)—C₂-C₄ alkene unsubstituted or substituted with halogen,—CH(X′)—C₂-C₄ alkyne unsubstituted or substituted with halogen,—CH═C(R¹⁰)(R¹¹), —C(CH₃)═C(R¹²)(R¹³), —C(R¹⁴)═C═C(R¹⁵)(R¹⁶), —C₃carbocycle unsubstituted or substituted with methyl or halogen,—CH(X′)—C₃ carbocycle unsubstituted or substituted with methyl orhalogen, C₃ heterocycle unsubstituted or substituted with methyl orhalogen, —CH(X′)—C₃ heterocycle unsubstituted or substituted with methylor halogen, —CH₂N₃, —CH₂CH₂N₃, and benzyloxymethyl, or R⁵ is a smallelectron withdrawing group; wherein R¹⁰ is hydrogen or C₁-C₃ alkylunsubstituted or substituted with halogen; R¹¹ is C₁-C₃ alkylunsubstituted or substituted with halogen; R¹² is hydrogen, halogen orC₁-C₃ alkyl unsubstituted or substituted with halogen; R¹³ is hydrogen,or C₁-C₃ alkyl unsubstituted or substituted with halogen; R¹⁴ ishydrogen or methyl; R¹⁵ and R¹⁶ are independently selected fromhydrogen, methyl and halogen; X is halogen; X′ is halogen or hydrogen;andadditionally, each of R¹, R², R^(2a), R³ and R^(3a) are optionallyhydrogen; optionally two R¹, R², R^(2a), R³ and R^(3a) on adjacentcarbon atoms are combined to form a double bond between said adjacentcarbon atoms; andprovided that at least one of R¹, R², R^(2a), R³, R^(3a), R⁴ and R⁵ is asmall electron withdrawing group, or R⁵ comprises a halogen, site ofunsaturation, carbocycle, heterocycle or azide, except when (i) R² andR^(2a) are both hydrogen, (ii) R³ and R^(3a) are both hydrogen, (iii) R¹is hydrogen, (iv) a double bond is present between said adjacent carbonatoms, or (v) R⁵ is benzyloxymethyl; andwherein protein, antibody or antibody derivative produced in vivo hasreduced fucosylation compared to the protein, antibody or antibodyderivative produced in vivo in the absence of the fucose analog.

In some embodiments of formulae (V) and (VI), R^(2a) and R^(3a) are eachhydrogen.

In some embodiments of formulae (V) and (VI), R⁵ is selected from thegroup consisting of —CH₃, —CH₂CH₃, —CH₂C≡CH, —CH═CHCH₃, -cyclopropyl,-oxirane, -oxirane substituted with methyl, —CH₂F, —CH₂Cl, —CH₂Br,—CH₂I, —CHF₂, —CH═C═CH₂, —CH₂N₃ and —CH₂CH₂N₃.

In some embodiments of formulae (V) and (VI), the small electronwithdrawing group is selected from fluoro, chloro, bromo, —CHF₂,—CH═C═CH₂, —C≡CH, —C≡CCH₃, —CH₂C≡CH, —CO₂H, —C(O)OC₁-C₄ alkyl,—CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is Br, Cl or I), andmethoxiran.

In some embodiments of formulae (V) and (VI), R⁵ is selected from thegroup consisting of —CH₃, —C≡CH, —CH₂F, —CH₂Br, and —CHF₂. In somefurther embodiments, each of R¹, R², R^(2a), R³, R^(3a) and R⁴ isindependently selected from the group consisting of —OH, —OC(O)H, and—OC(O)C₁-C₁₀ alkyl.

In some embodiments of formulae (V) and (VI), the small electronwithdrawing group is selected from fluoro, chloro, bromo, —CHF₂,—CH═C═CH₂, —C≡CH, —C≡CCH₃, —CH₂C≡CH, —CO₂H, —C(O)OC₁-C₄ alkyl,—CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is Br, Cl or I), andmethoxiran.

In some embodiments of formulae (V) and (VI), at least two of R¹, R²,R³, R^(3a) and R⁴ are independently selected small electron withdrawinggroups.

In some embodiments of formulae (V) and (VI), the fucose analog isselected from compounds of Tables 1, 2 or 3.

Pharmaceutical Compositions

Fucose, analogs of formulae I, II, III, IV, V and VI (hereinafter‘fucose analogs’) can be formulated for therapeutic applications. Thefucose analogs can be formulated as pharmaceutical compositionscomprising a therapeutically or prophylactically effective amount of thefucose analog and one or more pharmaceutically compatible (acceptable)ingredients. For example, a pharmaceutical or non-pharmaceuticalcomposition typically includes one or more carriers (e.g., sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like). Water is a more typical carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions,Suitable excipients include, for example, amino acids, starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol, and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions willtypically contain a therapeutically effective amount of the fucoseanalog, typically in purified form, together with a suitable amount ofcarrier so as to provide the form for proper administration to thepatient. The formulations correspond to the mode of administration.

The pharmaceutical compositions described herein can be in any form thatallows for the composition to be administered to an animal (e.g., amammal). The pharmaceutical compositions described herein can be in anyform that allows for the composition to be administered to a mammal. Thepharmaceutical compositions described herein can be in any form thatallows for the composition to be administered to a human.

The compositions can be in the form of a solid or liquid. Typical routesof administration include, without limitation, oral, parenteral,sublingual, and ocular. Parenteral administration includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. Preferably, the compositions are administeredparenterally or orally. These pharmaceutical compositions can beformulated so as to allow a fucose analog to be bioavailable uponadministration of the composition to an animal. Compositions can alsotake the form of one or more dosage units, where for example, a tabletcan be a single dosage unit, and a container of a fucose analog in solidform can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the fucose analog, the manner ofadministration, the composition employed, and the severity of thedisease or condition being treated.

The pharmaceutically acceptable carrier or vehicle can be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid.

When intended for oral administration, the composition is preferably insolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: hinders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such asstarch, lactose or dextrins, disintegrating agents such as alginic acid,sodium alginate, Primogel, corn starch and the like; lubricants such asmagnesium stearate or Sterotex; glidants such as colloidal silicondioxide; sweetening agents such as sucrose or saccharin, a flavoringagent such as peppermint, methyl salicylate or orange flavoring, and acoloring agent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it can contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, a composition can comprise one or more of a sweeteningagent, preservatives, dye/colorant and flavor enhancer. In a compositionfor administration by injection (as described above), one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent can also be included.

Liquid compositions, whether they are solutions, suspensions or otherlike form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. Physiological saline is a preferred adjuvant. Aninjectable composition is preferably sterile.

As noted above, the amount of the fucose analog that is effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro or in vivo assays canoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

The compositions comprise an effective amount of a fucose analog suchthat a suitable dosage will be obtained. Typically, this amount is atleast about 0.01% of a fucose analog by weight of the composition. Whenintended for oral administration, this amount can be varied to rangefrom about 0.1% to about 80% by weight of the composition. Preferredoral compositions can comprise from about 4% to about 50% of the fucoseanalog by weight of the composition. Preferred compositions of thepresent invention are prepared so that a parenteral dosage unit containsfrom about 0.01% to about 2% by weight of the fucose analog.

For intravenous administration, the composition can comprise from about1 to about 250 mg of a fucose analog per kg of the animal's body weight.In some embodiments, the amount administered will be in the range fromabout 1 to about 25 mg/kg of body weight of the fucose analog.Preferably, the amount administered will be in the range from about 4 toabout 25 mg/kg of body weight of the fucose analog.

Generally, the dosage of fucose analog administered to an animal istypically about 0.1 mg/kg to about 250 mg/kg of the animal's bodyweight. Preferably, the dosage administered to an animal is betweenabout 0.1 mg/kg and about 20 mg/kg of the animal's body weight, morepreferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.

The compositions comprise an effective amount of a fucose analog suchthat a suitable dosage will be obtained. Typically, this amount is atleast about 0.01% of a fucose analog by weight of the composition. Whenintended for oral administration, this amount can be varied to rangefrom about 0.1% to about 80% by weight of the composition. Preferredoral compositions can comprise from about 4% to about 50% of the fucoseanalog by weight of the composition. Preferred compositions of thepresent invention are prepared so that a parenteral dosage unit containsfrom about 0.01% to about 2% by weight of the fucose analog.

For intravenous administration, the composition can comprise from about1 to about 250 mg of a fucose analog per kg of the animal's body weight.In some embodiments, the amount administered will be in the range fromabout 1 to about 25 mg/kg of body weight of the fucose analog.Preferably, the amount administered will be in the range from about 4 toabout 25 mg/kg of body weight of the fucose analog.

Generally, a fucose analog or a pharmaceutical composition thereof canbe administered on a daily, weekly, biweekly or monthly schedule,according to the desired effect. A fucose analog or a pharmaceuticalcomposition thereof can be administered from about 1 to 5, about 1 toabout 10, about 1 to about 15, or more cycles, wherein each cycle is amonth in duration. The doses within each cycle can be given on daily,every other day, twice weekly, weekly, bi-weekly, once every three weeksor monthly. A cycle may optionally include a resting period, duringwhich fucosylation of proteins (e.g., antibodies or other proteins)increases. Alternatively, a resting period can be included betweencycles. Such a resting period can allow restoration of fucosylation ofproteins involved in essential functions.

The preferred mode of administration of a fucose analog, or apharmaceutical composition thereof, is left to the discretion of thepractitioner, and will depend in-part upon the site of the medicalcondition (such as the site of cancer or autoimmune disease). In oneembodiment, the fucose analog or compositions are administeredparenterally. In another embodiment, the fucose analog or compositionsare administered orally.

In specific embodiments, it can be desirable to administer one or morefucose analogs or compositions locally to the area in need of treatment.This can be achieved, for example, and not by way of limitation, bylocal infusion during surgery; topical application; by injection; or bymeans of an implant, the implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. In one embodiment, administration can be by direct injectionat the site (or former site) of a cancer, tumor or neoplastic orpre-neoplastic tissue.

In another embodiment, administration can be by direct injection at thesite (or former site) of a manifestation of an autoimmune disease.

In another embodiment, the fucose analogs can be delivered in a vesicle,in particular a liposome (see Langer, Science 249:1527-1533 (1990),Treat et al., in LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE ANDCANCER, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In yet another embodiment, the fucose analogs or compositions can bedelivered in a controlled release system. In one embodiment, a pump canbe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); CONTROLLED DRUGBIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). Other controlled-release systems discussed inthe review by Langer (Science 249:1527-1533 (1990)) can be used.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a fucose analog is administered. Such pharmaceutical carriers canbe liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. The carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. In addition, auxiliary, stabilizing, thickening,lubricating and coloring agents can be used. In one embodiment, whenadministered to an animal, the fucose analogs or compositions andpharmaceutically acceptable carriers are sterile. Water is a preferredcarrier when the fucose analogs are administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical carriers also include excipients such as starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The present compositions, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents.

Therapeutic Methods Using Fucose Analogs to Reduce Antibody and OtherProtein Fucosylation In Vivo

The fucose analogs of formulae I, II, III, IV, V and VI (hereinafter‘the fucose analogs’) as provided herein are useful for treating cancer,an autoimmune disease or an infectious disease in an animal.

Treatment of Cancer

The fucose analogs are useful for treating cancer in patients.Administration of is fucose analog to an animal (e.g., a mammal, such asa human) in need thereof can result in inhibition of the multiplicationof a tumor cell(s) or cancer cell(s), or treatment of cancer in ananimal (e.g., a human patient). The fucose analogs can be usedaccordingly a variety of settings for the treatment of animal cancers.

The fucose analogs are also useful for enhancing the in vivo productionof antibodies lacking core fucosylation. Increasing the proportion ofsuch antibodies against cancer targets in a patient can result ininhibition of the multiplication of a tumor cell(s) or cancer cell(s),or treatment of cancer in an animal (e.g., a human patient). The fucoseanalogs can be used accordingly in a variety of settings for thetreatment of animal cancers.

Particular types of cancers that can be treated with the fucose analogsinclude, solid tumors and hematologic malignancies. Such cancersinclude, but are not limited to: (1) solid tumors, including but notlimited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer,colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breastcancer, ovarian cancer, prostate cancer, esophogeal cancer, stomachcancer, oral cancer, nasal cancer, throat cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, uterine cancer, testicular cancer, small cell lung carcinoma,bladder carcinoma, lung cancer, epithelial carcinoma, glioma,glioblastoma, multiforme astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, skin cancer, melanoma,neuroblastoma, and retinoblastoma; (2) blood-borne cancers, includingbut not limited to acute lymphoblastic, leukemia “ALL”, acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”,acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairycell leukemia, multiple myeloma, acute and chronic leukemias, e.g.,lymphoblastic myelogenous and lymphocytic myelocytic leukemias, and (3)lymphomas such as Hodgkin's disease, non-Hodgkin's Lymphoma, multiplemyeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, andPolycythemia vera.

Multi-Modality Therapy for Cancer

Cancer, including, but not limited to, a tumor, a metastasis, or anydisease or disorder characterized by uncontrolled cell growth, can betreated or prevented by administration of a fucose analog of any offormulae I, II, III, IV, V or VI as provided above, to an animal (e.g.,a mammal, such as a human) in need thereof. In some embodiments, theinvention provides methods for treating or preventing cancer, comprisingadministering to an animal in need thereof an effective amount of afucose analog and optionally a chemotherapeutic agent. In one embodimentthe chemotherapeutic agent is that with which treatment of the cancerhas not been found to be refractory. In another embodiment, thechemotherapeutic agent is that with which the treatment of cancer hasbeen found to be refractory. The fucose analogs can be administered toan animal that has also undergone surgery as treatment for the cancer.

In one embodiment, the additional method of treatment is radiationtherapy.

In a specific embodiment, the fucose analog is administered concurrentlywith the chemotherapeutic agent or with radiation therapy. In anotherspecific embodiment, the chemotherapeutic agent or radiation therapy isadministered prior or subsequent to administration of a fucose analog,preferably at least an hour, five hours, 12 hours, a day, a week, twoweeks, three weeks, a month, or several months (e.g., up to threemonths), prior or subsequent to administration of a fucose analog.

A chemotherapeutic agent can be administered over a series of sessions,and can be any one or a combination of the chemotherapeutic agentsprovided herein. With respect to radiation, any radiation therapyprotocol can be used depending upon the type of cancer to be treated.For example, but not by way of limitation, x-ray radiation can beadministered; in particular, high-energy megavoltage (radiation ofgreater that 1 MeV energy) can be used for deep tumors, and electronbeam and orthovoltage x-ray radiation can be used for skin cancers.Gamma-ray emitting radioisotopes, such as radioactive isotopes ofradium, cobalt and other elements, can also be administered.

Additionally, the invention provides methods of treatment of cancer witha fucose analog as an alternative to chemotherapy or radiation therapy,where the chemotherapy or the radiation therapy has proven or can provetoo toxic, e.g., results in unacceptable or unbearable side effects, forthe subject being treated. The animal being treated can, optionally, betreated with another cancer treatment such as surgery, radiation therapyor chemotherapy, depending on which treatment is found to be acceptableor bearable.

Multi-Drug Therapy for Cancer

The present invention includes methods for treating cancer, comprisingadministering to an animal in need thereof an effective amount of afucose analog and a therapeutic agent that is an anti-cancer agent.Suitable anticancer agents include, but are not limited to,methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine,hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas,cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan,nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan,a camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin,dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferredembodiment, the anti-cancer agent includes, but is not limited to:alkylating agents, nitrogen mustards (cyclophosphamide, Ifosfamide,trofosfamide, Chlorambucil), nitrosoureas (carmustine (BCNU), Lomustine(CCNU)), alkylsulphonates (busulfan, Treosulfan), triazenes(Dacarbazine), platinum containing compounds (cisplatin, oxaliplatin,carboplatin), plant alkaloids (vinca alkaloids—vincristine, Vinblastine,Vindesine, Vinorelbine), taxoids (paclitaxel, Docetaxol), DNATopoisomerase Inhibitors, Epipodophyllins (etoposide, Teniposide,Topotecan, 9-aminocamptothecin, camptothecin), crisnatol, mitomycins(Mitomycin C); Anti-metabolites such as Anti-folates: DHFR inhibitors:methotrexate, Trimetrexate; IMP dehydrogenase Inhibitors: mycophenolicacid, Tiazofurin, Ribavirin, EICAR; Ribonuclotide reductase Inhibitors:hydroxyurea deferoxamine; Pyrimidine analogs: Uracil analogs:5-Fluorouracil, Floxuridine, Doxifluridine, Ratitrexed; Cytosineanalogs: cytarabine (ara C), Cytosine arabinoside, fludarabine; Purineanalogs: mercaptopurine, Thioguanine; Hormonal therapies: Receptorantagonists: Anti-estrogen: Tamoxifen, Raloxifene, megestrol; LHRHagonists: goscrclin, Leuprolide acetate; Anti-androgens: flutamide,bicalutamide; Retinoids/Deltoids: Vitamin D3 analogs: EB 1089, CB 1093,KH 1060; Photodynamic therapies: vertoporfin (BPD-MA), Phthalocyanine,photosensitizer Pc4, Demethoxy-hypocrellin A (2BA-2-DMHA); Cytokines:Interferon-alpha, Interferon-gamma; Tumor necrosis factor: Others:Isoprenylation inhibitors: Lovastatin; Dopaminergic neurotoxins:1-methyl-4-phenylpyridinium ion; Cell cycle inhibitors: staurosporine;Actinomycins: Actinomycin D, Dactinomycin; Bleomycins: bleomycin A2,Bleomycin B2, Peplomycin; Anthracyclines: daunorubicin, Doxorubicin(adriamycin), Idarubicin, Epirubicin, Pirarubicin, Zorubicin,Mitoxantrone; MDR inhibitors: verapamil; and Ca²⁺ ATPase inhibitors:thapsigargin.

Adjuvant Therapy for Cancer

The fucose analogs can be used as an adjuvant, in combination with acancer vaccine. The term “cancer vaccine” as used herein means acompound that selectively damages tumor cells by inducing and/orenhancing a specific immune response against the tumor cells. A cancervaccine can be, for example, a medicament comprising a peptide,polypeptide or protein of a TAA or TSA, and pharmaceutical compositionscontaining a peptide, polypeptide or protein of a TAA or TSA. As usedherein, TSA refers to a “tumor-specific antigen” and TAA refers to atumor-associated antigen. TSAs are molecules unique to cancer cells.TAAs are molecules shared, but differently expressed, by cancer cellsand normal cells.

The dosage of the cancer vaccine can be determined with appropriatemodifications according to the extent of stimulation of an immuneresponse against the vaccine. In general, it is between 0.01 and 100mg/day/adult human, or preferably between 0.1 and 10 mg/day/adult humanas an active principle. The cancer vaccine can be administered from onceevery few days to every few months. Administration can he carried outaccording to well-known methods for administrating a peptide,polypeptide or protein for medical use, such as subcutaneously,intravenously, or intramuscularly. In order to induce and/or enhance theimmune response during administration, the peptide, polypeptide orprotein can be used, in the presence or absence of an appropriateadjuvant, with or without linking to a carrier. The carrier is notlimited as long as it exerts no harmful effect by itself onto the humanbody and is capable of enhancing antigenicity; cellulose, polymericamino acids, albumin, and the like can be given as examples of carriers.Adjuvants can be those used in general for peptide vaccine inoculation,and a Freund incomplete adjuvant (FIA), aluminum adjuvant (ALUM),Bordetella pertussis vaccine, mineral oil, and the like can be given asexamples. In addition, the formulation can be suitably selected byapplying a suitable well-known method for formulating a peptide,polypeptide or protein.

Otherwise, an effective cancer vaccine effect can be obtained also bycollecting a fraction of mononuclear cells from the peripheral blood ofa patient, incubating them with the peptide, polypeptide or protein ofthe present invention, and then returning the fraction of mononuclearcells in which induction of CTL and/or activation of CTL was observed,into the blood of the patient. A fucose analog can be co-administeredduring or after re-administration of the mononuclear cells. Cultureconditions, such as mononuclear cell concentration, concentration of thepeptide, polypeptide or proteins, culture time, and the like, can bedetermined by simply repeating studies. A substance having a capabilityto enhance the growth of lymphocytes, such as interleukin-2, may beadded during culturing.

Treatment of Autoimmune Diseases

The fucose analogs are useful for modulating an autoimmune disease orfor treating an autoimmune disease, so as to decrease symptoms and/orthe autoimmune response. The fucose analogs can be used accordingly in avariety of settings for the treatment of an autoimmune disease in ananimal.

In one embodiment, the fucose analogs down-regulate or down-modulate anauto-immune antibody associated with a particular autoimmune disease.

Particular types of autoimmune diseases that can be treated with thefucose analogs include, but are not limited to, Th2-lymphocyte relateddisorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis,allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graftversus host disease); Th1 lymphocyte-related disorders (e.g., rheumatoidarthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome,Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis,Wegener's granulomatosis, and tuberculosis); activated Blymphocyte-related disorders (e.g., systemic lupus erythematosus,Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes); andthose disclosed below.

Active Chronic Hepatitis, Addison's Disease, Allergic Alveolitis,Allergic Reaction, Allergic Rhinitis, Alport's Syndrome, Anaphlaxis,Ankylosing Spondylitis, Anti-phosholipid Syndrome, Arthritis,Ascariasis, Aspergillosis, Atopic Allergy, Atropic Dermatitis, AtropicRhinitis, Behcet's Disease, Bird-Fancier's Lung, Bronchial Asthma,Caplan's Syndrome, Cardiomyopathy, Celiac Disease, Chagas' Disease,Chronic Glomerulonephritis, Cogan's Syndrome, Cold Agglutinin Disease,Congenital Rubella Infection, CREST Syndrome, Crohn's Disease,Cryoglobulinemia, Cushing's Syndrome, Dermatomyositis, Discoid Lupus,Dressler's Syndrome, Eaton-Lambert Syndrome, Echovirus Infection,Encephalomyelitis, Endocrine opthalmopathy, Epstein-Barr VirusInfection, Equine Heaves, Erythematosis, Evan's Syndrome, Felty'sSyndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy,Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis,Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease,Guillain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia,Henoch-Schonlein, Purpura Idiopathic Adrenal Atrophy, IdiopathicPulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases,Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, JuvenileDiabetes Mellitus (Type I), Lambert-Eaton Syndrome, Laminitis, LichenManus, Lupoid Hepatitis, Lupus Lymphopenia, Meniere's Disease, MixedConnective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis,Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, PsoriaticArthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome,Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome,Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, SystemicLupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis,Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic EpidermalNecrolysis Type B, Insulin Resistance Type I Diabetes Mellitus,Ulcerative Colitis, Uveitis Vitiligo, Waldenstrom's Macroglobulemia, andWegener's Granulomatosis.

Multi-Drug Therapy of Autoimmune Diseases

The present invention also provides methods for treating an autoimmunedisease, comprising administering to an animal (e.g., a mammal) in needthereof an effective amount of a fucose analog and optionally atherapeutic agent that known for the treatment of an autoimmune disease.In one embodiment, the anti-autoimmune disease agent includes, but isnot limited to cyclosporine, cyclosporine A, mycophenylate, mofetil,sirolimus, tacrolimus, enanercept, prednisone, azathioprine,methotrexate, cyclophosphamide, prednisone, aminocaproic acid,chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone,chlorambucil, danazol, bromocriptine, meloxicam or infliximab.

Treatment of Infectious Diseases

The fucose analogs are useful for enhancing an immune response thatresults in increased killing or inhibition of the multiplication of acell that produces an infectious disease or for treating an infectiousdisease. The fucose analogs can be used accordingly in a variety ofsettings for the treatment of an infectious disease in an animal.

In one embodiment, the fucose analogs enhance an immune response,resulting in kill or inhibit, or increased killing or inhibition, of themultiplication of cells that produce a particular infectious disease.

Particular types of infectious diseases that can be treated with thefucose analogs include, but are not limited to, (1) Bacterial Diseases:Diptheria, Pertussis, Occult Bacteremia, Urinary Tract Infection,Gastroenteritis, Cellulitis, Epiglottitis, Tracheitis, AdenoidHypertrophy, Retropharyngeal Abcess, Impetigo, Ecthyma, Pneumonia,Endocarditis, Septic Arthritis, Pneumococcal, Peritonitis, BactermiaMeningitis, Acute Purulent Meningitis, Urethritis, Cervicitis,Proctitis, Pharyngitis, Salpingitis, Epididymitis, Gonorrhea, Syphilis,Listeriosis, Anthrax, Nocardiosis, Salmonella, Typhoid Fever, Dysentery,Conjuntivitis, Sinusitis, Brucellosis, Tullaremia, Cholera, BubonicPlague, Tetanus, Necrotizing Enteritis, Actinomycosis Mixed AnaerobicInfections, Syphilis, Relapsing Fever, Leptospirosis, Lyme Disease, RatBite Fever, Tuberculosis, Lymphadenitis, Leprosy, Chlamydia, ChlamydialPneumonia, Trachoma, Inclusion Conjunctivitis, Systemic; (2) FungalDiseases: Histoplamosis, Coccicidiodomycosis, Blastomycosis,Sporotrichosis, Cryptococcsis, Systemic Candidiasis, Aspergillosis,Mucormycosis, Mycetoma, Chromomycosis; (3) Rickettsial Diseases: Typhus,Rocky Mountain Spotted Fever, Ehrlichiosis, Eastern Tick-BorneRickettsioses, Rickettsialpox, Q Fever, and Bartonellosis; (4) ParasiticDiseases: Malaria, Babesiosis, African Sleeping Sickness, Chagas'Disease, Leishmaniasis, Dum-Dum Fever, Toxoplasmosis,Meningoencephalitis, Keratitis, Entamebiasis, Giardiasis,Cryptosporidiasis, Isosporiasis, Cyclosporiasis, Microsporidiosis,Ascariasis, Whipworm Infection, Hookworm infection, ThreadwormInfection, Ocular Larva Migrans, Trichinosis, Guinea Worm Disease,Lymphatic Filariasis, Loiasis, River Blindness, Canine HeartwormInfection, Schistosomiasis, Swimmer's Itch, Oriental Lung Fluke,Oriental Liver Fluke, Fascioliasis, Fasciolopsiasis, Opisthorchiasis,Tapeworm Infections, Hydatid Disease, Alveolar Hydatid Disease; (5)Viral Diseases: Measles, Subacute sclerosing panencephalitis, CommonCold, Mumps, Rubella, Roseola, Fifth Disease, Chickenpox, Respiratorysyncytial virus infection, Croup, Bronchiolitis, InfectiousMononucleosis, Poliomyelitis, Herpangina, Hand-Foot-and-Mouth Disease,Bornholm Disease, Genital Herpes, Genital Warts, Aseptic Meningitis,Myocarditis Pericarditis, Gastroenteritis, Acquired ImmunodeficiencySyndrome (AIDS), Reye's Syndrome, Kawasaki Syndrome, Influenza,Bronchitis, Viral “Walking” Pneumonia, Acute Febrile RespiratoryDisease, Acute pharyngoconjunctival fever, Epidemickeratoconjunctivitis, Herpes Simplex Virus 1 (HSV-1), Herpes SimplesVirus 2 (HSV-2), Shingles, Cytomegalic. Inclusion Disease, Rabies,Progressive Multifocal Leukoencephalopathy, Kuru, Fatal FamilialInsomnia, Creutzfeldt-Jakob Disease, Gerstmann-Straussler-ScheinkerDisease, Tropical Spastic Paraparesis, Western Equine Encephalitis,California Encephalitis, St. Louis Encephalitis, Yellow Fever, DengueLymphocytic choriomeningitis, Lassa Fever, Hemorrhagic Fever, Hantvirus,Pulmonary Syndrome, Marburg Virus Infections, Ebola Virus Infections andSmallpox.

Multi-Drug Therapy of Infectious Diseases

The present invention also provides methods for treating an infectiousdisease, comprising administering to an animal (e.g., a mammal) in needthereof a fucose analog and optionally a therapeutic agent that is ananti-infectious disease agent. In one embodiment, the anti-infectiousdisease agent is, but not limited to: (1) Antibacterial Agents: β-LactamAntibiotics: Penicillin G, Penicillin V, Cloxacilliin, Dicloxacillin,Methicillin, Nafcillin, Oxacillin, Ampicillin, Amoxicillin,Bacampicillin, Azlocillin, Carbenicillin, Mezlocillin, Piperacillin,Ticarcillin; Aminoglycosides: Amikacin, Gentamicin, Kanamycin, Neomycin,Netilmicin, Streptomycin, Tobramycin; Macrolides: Azithromycin,Clarithromycin, Erythromycin, Lincomycin, Clindamycin; Tetracyclines:Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline;Quinolones: Cinoxacin, Nalidixic Acid, Fluoroquinolones: Ciprofloxacin,Enoxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Norfloxacin,Ofloxacin, Sparfloxacin, Trovafloxicin; Polypeptides: Bacitracin,Colistin, Polymyxin B; Sulfonamides: Sulfisoxazole, Sulfamethoxazole,Sulfadiazine, Sulfamethizole, Sulfacetamide; Miscellaneous AntibacterialAgents: Trimethoprim, Sulfamethazole, Chloramphenicol, Vancomycin,Metronidazole, Quinupristin, Dalfopristin, Rifampin, Spectinomycin,Nitrofurantoin; Antiviral Agents: General Antiviral Agents: Idoxuradine,Vidarabine, Trifluridine, Acyclovir, Famcicyclovir, Pencicyclovir,Valacyclovir, Gancicyclovir, Foscarnet, Ribavirin, Amantadine,Rimantadine, Cidofovir; Antisense Oligonucleotides; Immunoglobulins;Inteferons; Drugs for HIV infection: Zidovudine, Didanosine,Zalcitabine, Stavudine, Lamivudine, Nevirapine, Delavirdine, Saquinavir,Ritonavir, Indinavir and Nelfinavir.

Other Therapeutic Agents

The present methods can further comprise the administration of a fucoseanalog and a therapeutic agent or pharmaceutically acceptable salts orsolvates thereof. The fucose analog and the therapeutic agent can actadditively or, more preferably, synergistically. In a preferredembodiment, a composition comprising a fucose analog is administeredconcurrently with the administration of one or more therapeuticagent(s), which can be part of the same composition or in a differentcomposition from that comprising the fucose analog. In anotherembodiment, a fucose analog is administered prior to or subsequent toadministration of the therapeutic agent(s).

In the present methods for treating cancer, an autoimmune disease or aninfectious disease, the therapeutic agent also can be an antiemeticagent. Suitable antiemetic agents include, but are not limited to,metoclopromide, domperidone, proclorperazine, promethazine,chlorpromazine, trimethobenzamide, ondansetron, granisetron,hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron,benzquinamide, bietanautine, bromopride, buclizine, clebopride,cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine,methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine,sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine andtropisetron.

In another embodiment, the therapeutic agent can be an hematopoieticcolony stimulating factor. Suitable hematopoietic colony stimulatingfactors include, but are not limited to, filgrastim, sargramostim,molgramostim and erythropoietin alfa.

In still another embodiment, the therapeutic agent can be an opioid ornon-opioid analgesic agent. Suitable opioid analgesic agents include,but are not limited to, morphine, heroin, hydromorphone, hydrocodone,oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine,buprenorphine, meperidine, lopermide, anileridine, ethoheptazine,piminidine, betaprodine, diphenoxylate, fentanil, sufentanil,alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine,pentazocine, cyclazocine, methadone, isomethadone and propoxyphene.Suitable non-opioid analgesic agents include, but are not limited to,aspirin, celecoxib, rofecoxib, diclofinac, etodolac, fenoprofen,flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac,meclofenamate, mefenamic acid, nabumetone, naproxen, piroxicam andsulindac.

The invention is further described in the following examples, which arenot intended to limit the scope of the invention.

EXAMPLES Example 1 In Vivo Production Of Non-Fucosylated AntibodiesMethods:

Female BALB/c mice were immunized with Keyhole Limpet Hernocyanin (KLH)30-60 days prior to starting this study. For the first study, mice weredosed ip with 150 mg/kg of alkynyl fucose (SGD-1887), alkynyl fucoseperacetate (SGD-1890), 2-fluorofucose (SGD-2083), or 2-fluorofucosetriacetate (SGD-2084) daily for 7 days or were untreated. On day 2, micewere also boosted with KLH. On the day after the last ip dose, mice wereterminally bled and serum obtained. For the second study, mice weregiven 100 mM of 2-fluorofucose (SGD-2083) in their drinking water orgiven untreated water for 7 days, boosted with KLH, and continued with100 mM 2-fluorofucose containing drinking water or untreated water for 7more days before being terminally bled. Serum was then obtained. No micedied in either study. For both studies, serum was passed over acommercial anti-KLH affinity column to obtain KLH-specific polyclonalantibodies. The flow through from the anti-KLH affinity column waspassed over a commercial protein A column to obtain the remainingantibodies.

Dot blots: 0.5 μg each of antibodies from untreated and treated animals,as well as standards of antibody cAC10 having known amounts of corefucosylation (0 to 100% fucose), were blotted onto a nitrocellulosemembrane. The proteins levels were visualized by Ponceau staining. Theblot was probed with biotinylated Aspergillus oryzae L-fucose-specificlectin (AOL) (which binds to fucosylated antibodies) and developed withstreptavidin HRP and ECL. Gel loading (visible) and fucose signals(bioluminescence) were measured with an Alpha Innotech camera andquantitated with the machine software.

Gas chromatography (GC): 40 μg each of the antibodies from untreated andtreated animals that had been dialyzed against water, were subjected tomethanolysis in methanolic HCl. Control samples of antibody cAC10 with 0to 100% fucose were similarly treated. The resulting methylglycosideswere derivatized by trimethylsilylation of the monosaccharide alcoholsusing a commercially available cocktail, Tri-Sil. The resultingtrimethylsilyl methylglycosides were examined on a Hewlet Packard gaschromatograph with flame ionization detection using a temperaturegradient on an Agilent J&W DB-1 column. The relevant peaks wereidentified by retention time comparison to sugar standards derivatizedin parallel with the Ab samples. Peaks were integrated using the GCsoftware. The fucose/mannose peak area ratios were used to determine thefucosylation state of the antibodies.

Results:

Study 1: FIG. 1 shows the dot blot (left side) from antibodies that didnot bind to KLH, but were recovered from protein A column. Results areshown for cAC10 standards (lower dot blot, left most dashed rectangleand corresponding columns of upper dot blot), untreated control (lowerdot blot, second dashed rectangle from the left and corresponding columnof upper dot blot), and alkynyl fucose (SGD-1887; lower dot blot, middledashed rectangle and corresponding column of upper dot blot), alkynylfucose peracetate (SGD-1890, lower dot blot, second dashed rectanglefrom the right and corresponding column of upper dot blot), and2-fluorofucose (SGD-2083; lower dot blot, right most rectangle andcorresponding column of upper dot blot). Normalizing for loading level,the percent fucosylation is also shown in the graph on the right. Onaverage, the fucosylation levels of the antibodies were reduced by aboutone third, as compared to the untreated controls.

FIG. 2 shows the fucosylation levels of both the anti-KLH antibodies(panels A and B) and the remaining serum IgG molecules isolated from thetreated groups (panels C and D). Fucosylation levels are shown both aspercent fucosylation based on the cAC10 antibody standards (panels A andC) and the average value for the treated groups as a percentage of theaverage value for the untreated control group (panels B and D). Onaverage, the fucosylation levels of the anti-KLH antibodies were reducedby about one half by treatment with three of the fucose analogs. Theremaining collected antibodies also exhibited a reduction in corefucosylation of about one quarter. In this study, overall antibodylevels (KLH-specific and non-specific) increased in the mice afterexposure to KLH. As a result, most antibodies were newly synthesizedduring the treatment periods. These observations indicate that newlyproduced antibodies can exhibit reduced core fucosylation followingadministration of a fucose analog.

Study 2: in this study, the effect of oral administration of fucoseanalogs was examined FIG. 3 shows the results of treatment of mice byoral administration of fucose analogs. Antibody fucosylation levelsexamined were those of the antibodies that did not bind to KLH, but wererecovered from the protein A column. Results are shown for cAC10standards (upper and lower dot blots, left most rectangle), untreatedcontrol (upper and lower dot blots, second from the left (upper) andright rectangles), and 2-fluorofucose (upper and lower dot blots, secondfrom the left (lower) and second from the right rectangles (upper andlower)). Normalizing for loading level, the percent fucosylation is alsoshown in the graph at the right. Core fucosylation levels of antibodiesfrom the treated animals were nearly eliminated: on average,fucosylation levels were 7% for treated and 81% for untreated animals.These observations indicate that oral administration of fucose analogsis an effective means to decrease antibody fucosylation levels.

Example 2 Activity of Fucose Analogs In Vitro in Cell Culture

Fucose analogs have been evaluated for their effect on antibody corefucosylation at concentrations of 50 μM and 1 mM generally as describedin Published US Patent Application 2009-0317869. Briefly, the protocolwas as follows: A CHO DG44 cell line producing a humanized IgG1anti-CD70 monoclonal antibody, h1F6 (see International PatentPublication WO 06/113909) was cultured at 7.5×10⁵ cells per mL in 2 mLsof CHO culture media at 37°, 5% CO₂ and shaking at 100 RPM in a 6 welltissue culture plate. Media was supplemented with insulin like growthfactor (IGF), penicillin, streptomycin and either 1 mM or 50 μM of thefucose analog. On day 5 post inoculation, the culture was centrifuged at13000 RPM for 5 minutes to pellet the cells; antibodies were thenpurified from supernatant.

Antibody purification was performed by applying the conditioned media toprotein A resin pre-equilibrated with 1× phosphate buffered saline(PBS), pH 7.4. After washing resin with 20 resin bed volumes of 1×PBS,antibodies were eluted with 5 resin bed volumes of Immunopure IgGelution buffer (Pierce Biotechnology, Rockford, Ill.). A 10% volume of1M Tris pH 8.0 was added to neutralize the eluted fraction. The resultsare shown in the following tables.

TABLE 1 Name Inhibition Inhibition (Chemical name) R⁵ R¹-R⁴ at 50 μM at1 mM Alkynyl fucose —C≡CH —OH >80% ND (5-ethynylarabinose) Alkynylfucose peracetate —C≡CH —OAc >80% >80% Alkynyl fucose tetraacetate(5-ethynylarabinose tetraacetate) 5-propynyl fucose tetraacetate —C≡CCH₃—OAc   50% >80% (5-propynylarabinose tetraacetate) propargyl fucosetetraacetate —CH₂C≡CH —OAc ~10% ~10-20% ((3S,4R,5R,6S)-6-(prop-2-ynyl)-tetrahydro-2H-pyran-2,3,4,5- tetrayl tetraacetate Peracetyl galactose—OAc —OAc  ~0%  ~0% (galactose pentaacetate) 5-vinyl fucose tetraaceate—CHCH₂ —OAc  ~0%  ~4% (5-ethylenylarabinose tetraacetate) 6-cyano fucosetetraacetate —CH₂CN —OAc   30% >80% (6-cyanofueose tetraacetate) 5-cyanofucose tetraacetate —CN —OAc   20% ND (pyranose form)(5-cyanoarabinopyranose tetraacetate) 5-cyano fucose tetraacetate —CN—OAc 5-10% ND (furanose form) (5-cyanoarabinofuranose tetraacetate)5-methylester fucose —C(O)OCH₃ —OAc   30% >80% tetraacetate(5-carboxymethyl arabinose tetraacetate) 5-(CH(OAc)CH₃) peracetyl—CH(OAc)CH₃ —OAc  ~0%   40% fucose (6-methylgalactose pentaacetate)5-methyloxiran-arabinose tetraacetate ((3S,4R,5S,6R)-6-((S)-2-methyloxiran-2-yl)-tetrahydro- 2H-pyran-2,3,4,5-tetrayl tetraacetate)

—OAc  ~0% ~35-40% 6-iodo-fucose tetraacetate —CH₂I —OAc    3%   30%(6-iodofucose tetraacetate) 6-chloro-fucose tetraacetate —CH₂Cl —OAc  20% 20-30% (6-chlorofucose tetraacetate 6-bromo-fucose tetraacetate—CH₂Br —OAc   50%   80% (6-bromafucose tetraacetate) Alkynyl fucosetetrapropanonate —C≡CH —OC(O)CH₂—CH₃ >80% >80% (5-ethynylarabinosetetrapropropanoate) Alkynyl fucose tetra-n- —C≡CH—OC(O)CH₂)₄—CH₃ >80% >80% hexanoate (5-ethynylarabinose tetrahexanoate)Alkynyl fucose —C≡CH —OC(O)C(CH₃)₃   20% 60% tetrakis(trimethylacetate)(5-ethynylarabinose tetra(trimethylacetate)) Alkynyl fucose —C≡CH—OC(O)C(CH₃)₃    5% 10% tetrakis(trimethylacetate) (5-ethynylarabinosetetra(trimethylacetate)) Alkynyl fucose 1, 2, 3- —C≡CH —OC(O)C(CH₃)₃ ~0% ND (trimethylacetate) and —OH (5-ethynylarabinose 1, 2, 3-(trimethylacetate)) Alkynyl fucose —C≡CH —OC(O)C(CH₃)₃ >80% NDdi(trimethylacetate) and —OH (5-ethynylarabinose 1, 3-(trimethylacetate)) Alkynyl fucose pernicotinate —C≡CH—C(O)-3-pyridyl >80% >80% Alkynyl fucose perisonicotinate —C≡CH—C(O)-4-pyridyl >80% >80% Alkynyl fucose per-PEG ester —C≡CH—C(O)— >80% >80% (CH₂CH₂O)₂— OCH₃ 1-methyl-2,3,4-triacetyl alkynyl —C≡CHR¹ = OCH₃   68% >80% fucose R², R³, R⁴ = OAc Alkynyl fucoseperisobutanoate —C≡CH —OC(O)CH(CH₃)₂ >80% >80% “ND” means not detecteddue to poor antibody production or inhibition of cell growth in thepresence of the fucose analog.

TABLE 2 Inhibition Inhibition Name at 50 at 1 (Chemical name) R⁵ R¹R²/R^(2a) R³/R^(3a) μM mM 2-deoxy-2-fluorofucose —CH₃ —OH —F/—H—OAc/ >80% >80% diacetate —H (R⁴ = OAc) 2-deoxy-2-chlorofucose —CH₃ —OAc—Cl/ —OAc/ 17% >80% triacetate —H —H (R⁴ = OAc) Allene —CH=C=CH₂ —OAc—OAc/ —OAc/ 23% 34% (R⁴ = OAc) —H —H 2-deoxy-2-fluorofucose —CH₃ —OH—F/—H —OH/ >80% >80% (R⁴ = OH) —H 2-deoxy-2-fluorofucose —CH₃ —OAc —F/—H—OAc/ >80% >80% peracetate —H (R⁴ = OAc) 1,2-difluoro-1,2-didexoy —CH₃—F —F/—H —OAc/ >80% >80% fucose peracetate —H (R⁴ = OAc)6,6-difluorofucose —CHF₂ —OAc —OAc/ —OAc/ >80% >80% tetraacetate —H —H(R⁴ = OAc) 2-deoxy-2,2- —CH₃ —OAc —F/—F —OAc/ 0 64% difluorofucopyranose—H triacetate (alpha) (R⁴ = OAc) 2-deoxy-2,2- —CH₃ —OAc —F/—F —OAc/ 075% difluorofucopyranose —H triacetate (beta) (R⁴ = OAc)6-methyl-tetrahydro-2H- —CH₃ —OAc —H/—H —OAc/ 0 36% pyran-2,4,5-triyltriacetate —H (R⁴ = OAc) 5-Benzyloxy fucose —CH₂OCH₂Ph —OAc —OAc/ —OAc/0 75% peracetate —H —H (R⁴ = OAc) “ND” not detected due to poor antibodyproduction or inhibition of cell growth in the presence of the fucoseanalog.

Certain other fucose analogs were tested for their ability to beincorporated into antibodies. These fucose analogs were tested atconcentrations of 50 μM and 1 mM using the methodology as describedabove. The results are shown in the following table.

TABLE 3 Name % Incor- (Chemical name) R⁵ R¹-R⁴ poration Propargyl fucoseor (3S,4R,5R)-6-(prop-2- ynyl)tetrahydro-2H-pyran- 2,3,4,5-tetrayltetraacetate

—OAc   80% (1 mM) 5-(Z)-propenyl fucose peracetate

—OAc ~30% Isopropenyl peracetyl fucose or (3S,4R,5R,6S)-6-(prop-1-en-2-yl)-tetrahydro-2H-pyran-2,3,4,5- tetrayl tetraacetate

—OAc >80% (1 mM and 50 uM) 5-ethyl fucose —CH₂CH₃ —OH >80% or (1 mM and(3S,4R,5S,6S)-6-ethyl- 50 uM) tetrahydro-2H-pyran-2,3,4,5- tetraol5-ethyl fucose peracetate —CH₂CH₃ —OAc >90% or (1 mM and(3S,4R,5S,6S)-6-ethyl- 50 uM) tetrahydro-2H-pyran-2,3,4,5- tetrayltetraacetate 5-cyclopropyl fucose or (3S,4R,5S,6S)-6-cyclopropyltetrahydro-2H- pyran-2,3,4,5-tetraol

—OH ~80% 5-cyclopropyl fucose peracetate or (3S,4R,5S,6S)-6-cyclopropyltetrahydro-2H- pyran-2,3,4,5-tetrayl tetraacetate

—OAc ~80% 5-propyloxyarabinose tetraacetate or (3S,4R,5S,6S)-6-((S)-2-methyloxiran-2-yl)tetrahydro- 2H-pyran-2,3,4,5-tetrayl tetraacetate

—OAc ~60% Fluoromethylene fucose —CH₂F —OAc >90% or (1 mM and(3S,4R,5S)-6- 50 μM) (fluoromethyl)tetrahydro-2H- pyran-2,3,4,5-tetrayltetraacetate 5-chloromethylene peracetyl —CH₂Cl —OAc ~80% fucose or(3S,4R,5S)-6- (chloromethyl)tetrahydro-2H- pyran-2,3,4,5-tetrayltetraacetate 5-bromomethylene peracetyl —CH₂Br —OAc ~50% fucose or(3S,4R,5S)-6- (50 uM; ) (bromomethyl)tetrahydro-2H- 20% atpyran-2,3,4,5-tetrayl tetraacetate 1 mM) 5-iodomethylene-peracetyl —CH₂I—OAc ~30% fucose or (3S,4R,5S)-6- (iodomethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate Azido peracetyl fucose —CH₂N₃ —OAc  60% or (3S,4R,5R)-6- (azidomethyl)tetrahydro-2H- pyran-2,3,4,5-tetrayltetraacetate 5-(2-azidoethyl) arabinose —CH₂CH₂N₃ —OAc   20%tetraacetate or (3S,4R,5R,6S)-6-(2- (azidoethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate —CH═C═CH₂  OAc ~30% Isopropylperacetyl fucose Isopropyl  OAc Not or detected (3S,4R,5R,6S)-6-isopropyltetrahydro-2H-pyran- 2,3,4,5-tetrayl tetraacetate

These assays identified candidate compounds for inhibition of coreantibody fucosylation in mammals.

Example 3 Production of Non-Fucosylated Antibodies In Vivo FollowingOral Administration

In this study, the effects of oral administration of the fucose analog2-fluorofucose (SGD-2083) were further examined. Female BALC/c mice wereoffered 1, 10 and 100 mM 2-fluorofucose in their drinking water for 14days. Mice were immunized with TiterMAX Classic and offered 1, 10 and100 mM 2-fluorofucose in their drinking water for an additional 7 days.Mice were then terminally bled and serum obtained. Endogenous antibodieswere purified by passing the serum over a protein A column

The collected antibodies were evaluated for fucosylation levels by dotblot as follows. Antibodies from untreated and treated animals (0.5 μgeach), as well as standards of cAC10 with 0 to 100% fucose (only study1), were blotted onto a nitrocellulose membrane. The proteins levelswere visualized with Ponceau S. The blot was probed with biotinylatedAOL lectin and developed with streptavidin HRP and ECL (as describedabove). Gel loading (visible) and fucose signals (bioluminescence) weremeasured with an Alpha Innotech camera and quantitated with the machinesoftware.

Results:

There was a dose-dependent decrease in fucosylation levels of antibodiesover the three concentrations of 2-fluorofucose (SGD-2083). Referring toFIG. 4, antibody fucosylation levels were highest in the untreatedcontrol and 1 mM 2-fluorofucose groups (left and middle panels, uppertwo rectangles). Antibodies from the intermediate concentration of2-fluorofucose were nearly as depleted of fucose as the highconcentration of 100 mM 2-fluorofucose. These results confirm thatadministration of 2-fluorofucose to mice can inhibit core antibodyfucosylation.

Example 4 Affects of Fucose Analogs on Human Cells

The ability of different fucose analogs to inhibit the fucosylation ofIgG antibodies produced by human myeloma cells as well as thefucosylation of surface proteins of human cancer cell lines wasinvestigated. In a first study, the ability of fucose analogs to inhibitfucosylation of IgG produced by the cell line LP-1, a human multiplemyeloma cell line, was investigated. The antibodies produced by, andfound in the culture medium of, untreated. LP-1 cells were confirmed tobe of the IgG type by western blot using anti-human IgG detection (datanot shown). This was accomplished by growing 20 mL of LP-1 cells in aT-75 culture flask (250,000 cells/mL) for 5 days at 37° C. in ahumidified atmosphere of 5% CO₂ in IgG-depleted tissue culture media(90% RPMI with 10% IgG-depleted heat inactivated FBS). Harvest of thecells was by centrifugation (200×g, 4° C., 5 min), and the culturemedium was collected. The medium was filtered through a 0.22 μm filterand then incubated with 1 mL of a 50% MabSelect™ protein A resin slurryin PBS at 4° C. with rotation overnight to capture the IgG. The resinslurry was allowed to settle and most of the medium was removed. Theresin slurry was transferred with ˜0.5 mL media to two cellulose acetatefilter spin cups and was centrifuged at 5000×g for 1 min. The resin bedwas then washed 3 times with 0.5 mL PBS. The IgG was eluted with 700 μLof Pierce IgG elution buffer (into with 52 μL of 9 M Tris buffer, pH 9.5to adjust the pH after elution). The resulting elution was transferredto a 10,000 MW cutoff centrifugal concentrator and the sample wasconcentrated to approximately 20 μL. 1 μL of the concentrated sample wasloaded on an SDS polyacrylamide gel for separation followed by blottingonto nitrocellulose membrane. Staining of the blot for total protein waswith Ponceau S and for identification of isotype with anti-human IgGantibody. The total protein stain showed bands consistent with molecularweights of IgG heavy and light chains and the anti-human IgG stainshowed reaction with the protein band consistent with the molecularweight of heavy chain as expected.

Antibody fucosylation can also be determined using a biotin-labeledAspergillus oryzae L-fucose-specific lectin (AOL), which bindsspecifically to the α-1,6-linked fucose of the antibody. This method forfucose detection works for both blotted protein that has either beenseparated by SDS-PAGE or for protein that has been applied tonitrocellulose without separation. The fluorescent signal generatedusing the AOL-biotin conjugate with streptavidin-HRP binding and ECLdetection can be quantitated using an Alpha Innotech FlourChem® Qsystem. The IgG isolated from LP-1 culture displayed an AOL-dependentsignal in the band corresponding to the MW of the heavy chain asexpected (data not shown). The analogs 2-fluorofucose (SGD-2083) and2-fluorofucose peracetate (SGD-2084) did not inhibit fucosylation ofantibody, but alkynyl fucose peracetate, (SGD-1890) did.

To further evaluate the activities of different fucose analogs, 48different fucose analogs and other four glycosylation inhibitors weretested for their ability to affect the fucosylation the LP-1-generatedIgG. LP-1 cells (250,000 cells/mL, 3 mL per compound in 6-well plates)were incubated with 100 μM of each fucose analog for 5 days at 37° C.with a humidified atmosphere of 5% CO₂ in IgG-depleted tissue culturemedia (90% RPMI with 10% IgG-depleted heat inactivated FBS). The IgG wasisolated as described above using only 0.5 mL of MabSelect™ protein Aresin slurry and one spin cup per sample with elution in 400 μL of IgGelution buffer into 25 μL of 9 M Tris buffer, pH 9. The eluates wereconcentrated to 10-20 μL per sample and 2 μL of each of the concentratedeluates were dotted onto a nitrocellulose membrane and stained withPonceau S to estimate and adjust the sample loading for AOL staining.From this estimation of total protein in each sample, approximately 0.5μg of each sample was dotted onto the membrane, air-dried, and stainedwith Ponceau S. An image of this stained membrane was captured using anAlpha Innotech FlourChem® Q system. The membrane was then blocked with5% Bovine Serum Albumin (BSA) in Tris Buffered Saline (TBS) for 1 hr,washed with TBST (TBS with Triton) 3 times and then incubated with 5μg/ml biotinylated-AOL for 1 hr. The membrane was washed again with TBST3 times, followed by Streptavidin-HRP incubation for 30 min and finalwashes with TBST 3 times. The bioluminescent signal was revealed usingchemiluminescence reagents (ECL) and was analyzed using an AlphaInnotech FlourChem® Q system and Alphaview® software. The results areshown in the following table. For some analogs, multiple samples wereanalyzed, as indicated in the table.

TABLE 4 SCD % of control Molecule Namee number fucosylated IgG valuealkynyl fucose 1887 3 alkynyl fucose peracetate 1890 0; 0.08; 2 5-vinylfucose tetraacetate 1922 2 5-cyanomethylene fucose tetraacetate 1924 96L-galactono-1,4-lactone 1931 81 Methyl a-L-fucopyranoside 1932 875-propynyl fucose tetraacetate 1937 315 5-(Z)-propenyl fucose peracetate1944 72 6-propargylamino fucose 1950 40 5-methyl ester fucosetetraacetate 1959 94 castanospermine 1960 300 5-methylketo fucosetetraacetate 1964 5 6-bromo fucose tetraacetate 1969 1 5-isopropylfucose tetraaceate 1977 79 Kifunensine 1978 0.44; 2 propargyl fucosetetraacetate 1987 38 6-fluoro fucose tetraacetate 1988 15 5-ethyl fucosetetraacetate 1989 11 5-carboxamido fucose tetraacetate 1995 796-alkyne-6-acetoxy fucose tetraacetate 2004 29 alkynyl fucosetetrapropionate 2010 1.5 alkynyl fucose tetrahexanoate 2012 67 5-epoxyfucose tetraacetate 2020 5 6-thio galactose pentaacetate 2025 441-methyl fucose triacetate 2039 1 alkynyl fucose tetraisobutanoate 204334 6-formyl fucose tetraacetate 2045 70 6′6-difluoro fucose tetraacetate2046 2 alkynyl fucose tetranicotinate 2047 50 benzyloxy fucosetetraacetate 2048 114 alkynyl fucose tetra PEG ester 2057 64 alkynylfucose tetraisonicotinate 2058 34 1-methyl alkynyl fucose triacetate2059 89 6-carboxymethyl ester fucose tetraacetate 2061 71 6-keto-6-ethylfucose tetraacetate 2067 3 5-(2-cyanoethyl)arabinose tetraacetate 207051 D-galactose pentaacetate 2074 118 1,2-dideoxy-1,2-dehydro fucosediacetate 2080 159 1-deoxy fucose triacetate 2081 70 1,2-difuloro fucosediacetate 2082 87 2-fluoro-2-deoxy fucose 2083 66 2-fluoro-2-deoxyfucose tetraacetate 2084 52 6-allene fucose tetraacetate 2097 452-chloro-2-deoxy fucose tetraacetate 2099 146 2-deoxy fucose triacetate2108 104 3-thio fucose tetraacetate 2112 64 6-deoxy-L-talose 2113 934-deoxy fucose triacetate 2134 49

Three fucose analogs were chosen for a full SDS-PAGE/Western blotanalysis to show that changes in the fucose signal on the heavy chaincan be detected by this technique. The three analogs chosen were used ata concentration of 50 μM. These analyses compared the activity of2-fluorofucose (SGD-2083), 2-fluorofucose peracetate (SGD-2084), andalkynyl fucose peracetate (SGD-1890) with antibody from untreated cells.Use of alkynyl fucose peracetate produced IgG that did not showreactivity with the biotinylated AOL, confirming that changes in AOLsignal can be detected by this method while 2-fluorofucose (SGD-2083)and 2-fluorofucose peracetate (SGD-2084) showed no apparent change inthe AOL signal. These results are generally consistent with the resultsfor these compounds in Table 4.

Many of the fucose analogs tested in Table 4 appeared to decreasefucosylation of antibody produced by human myeloma cells. The dot blotsof the compounds showed that 10 of them were potentially stronginhibitors of IgG fucosylation in human cells, using decrease in AOLsignal as an indication of inhibition. These fucose analogs are alkynylfucose peracetate (SGD-1890), alkynyl fucose tetrapropionate (SGD-2010),1-methyl fucose triacetate (SGD-2039), 5-ethyl fucose tetraacetate(SGD-1989), 6-fluoro fucose tetraacetate (SGD-1988), 6-bromo fucosetetraacetate (SGD-1969), 6′6-difluoro fucose tetraacetate (SGD-2046),6-keto-6-ethyl fucose tetraacetate (SGD-2067), 5-epoxy fucosetetraacetate (SGD-2020), and 5-methylkao fucose tetraacetate (SGD-1964).

To further define the results of the AOL dot blot, samples of the IgGsproduced by cells treated with the following fucose analogs (that gavemoderate to strong decreases in the dot blot AOL signal) were isolatedand examined by reducing PLRP-MS to verify the fucosylation status usingthe MW of the heavy chain: alkynyl fucose peracetate; 5-vinylfucosetetraaceate; 5-methylketofucose tetraacetate; 6-bromofucosetetraacetate; 6-fluorofucose tetraacetate; 5-ethylfucose tetraacetate;5-epoxyfucose tetraacetate; 6′6-difluorofucose tetraacetate;6-keto-6-ethyl fucose tetraacetate; and 2-fluorofucose peracetate.

40 mL samples of LP-1 cells (250,000 cells/mL) were treated with 100 μMof a fucose analog for 5 days as described above, and the IgGs werepurified as described using protein A resin. The yields were estimatedby UV spectroscopy assuming an extinction coefficient of 1.4 AU/(mg/mL).Seven of the ten compounds yielded enough IgG to perform the analysis(use of SGD-2067, SGD-1964, and SGD-2020 yielded <10 μg of IgG, likelydue to toxicity of the analogs to the cells). The remaining IgGs werereduced with 10 mM DTT at 37° C. for 15 min and were separated on PLRPfollowed by MS analysis using a QTOF mass spectrometer. The resultingheavy chain peaks were examined and compared to the IgG generated byuntreated cells.

The mass spectrometry results are shown in Table 5 (below). The massspectrometry signals were evaluated by comparing the peak height of theheavy chain versus heavy chain minus fucose and heavy chain minus fucoseplus the mass of the fucose analog (which would arise if there wasincorporation of the analog into the antibody carbohydrate). Four of theten compounds tested were partial or full inhibitors of 1,6-fucosylationon the antibody. Alkynyl fucose peracetate (SGD-1890) provided completeinhibition while 2-fluorofucose peracetate (SGD-2084) was next best with70% inhibition followed by 6′6-difluorofucose tetraacetate (SGD-2046)and 6-bromofucose tetraacetate (SGD-1969) with ˜33 and 20% inhibitionmixed with incorporation of the analog into the carbohydrate.

TABLE 5 Results of PLRP-MS vs. dot blot for LP-1-generated IgG dot blotresults (% fucose signal SGD number PLRP/MS (inhibition orincorporation) of control) Untreated Control 100 SOD-1890 Full inhibitor~1 SOD-1922 Full incorporator 2 SOD-1964 Not determined 5 SOD-1969Partial incorporator and 20% fucose 1 inhibitor SOD-1988 Fullincorporator 15 SOD-1989 Fully incorporated 11 SOD-2020 Not determined 5SOD-2046 Partial incorporator and 33% fucose 2 inhibitor SOD-2067 Notdetermined 3 SOD-2084 70% inhibitor 52

Example 5 Affects of Fucose Analogs on Protein Fucosylation

The effects of the four partial to full inhibitors, alkynyl fucoseperacetate (SGD-1890), 2-fluorofucose peracetate (SGD-2084),6′6-difluorofucose tetraacetate (SGD-2046), and 6-bromofucosetetraacetate (SGD-1969), on protein cell surface fucosylation was testedfor human cancer cells by incubation of five different human-derivedcancer cell lines (Caki-1, PC-3, Ramos, LS174t, and HL60cy). 100 μM ofeach inhibitor was used under standard culture conditions forapproximately 1-2 weeks with regular changes of culture medium includingfresh inhibitor. After the incubation period, the cells were analyzed byFACS using four different detection reagents: biotinylated-Lensculimaris agglutinin-A (LCA), anti-Lewis^(x) antibody (anti-SSEA1), ananti-Lewis^(y) antibody (cBR96), and a Recombinant HumanP-Selectin/CD62P/Fc Fusion protein. The procedure involved washing ofthe cells with FACS buffer (PBS+10% bovine serum albumin+0.02% sodiumazide) 3 times followed by incubation with the primary detection reagentfor 1 hr at 4° C., followed by 3 washes with FACS buffer and thenincubation with the secondary detection reagent for 1 hr at 4° C. Thecells were finally washed with FACS buffer 3 times and resuspended inFACS buffer and examined using a BD FACScan instrument. The LCA reagentrecognizes sequences containing α-linked mannose residues and itsaffinity is markedly enhanced by α-linked fucose residues attached tothe N-acetylchitobiose portion of the core oligosaccharide. TheP-selectin fusion protein detects P-selectin ligand present on thesurface of cells, an interaction which involves the sialyl Lewis^(x)epitope present of the P-selectin ligand.

All of the cell lines examined showed staining with the LCA reagent,which recognizes sequences containing α-linked mannose residues, theaffinity of which is markedly enhanced by α-linked fucose residuesattached to the N-acetylchitobiose portion of the core oligosaccharide.The LCA detection of this sugar epitope was decreased upon treatment ofthe cells with all of the inhibitors (100 μM). This suggests that theoverall presence of fucose on the cell surface is affected by treatmentwith the six fucose analogs examined.

FIG. 7 shows the results of these studies. For Lewis^(x), of the celllines examined only untreated LS1745t and HL60cy had significantLewis^(X) detected on the cell surface (anti-SSEAI staining) (FIG. 7A).The anti-SSEAI detection of this structure was significantly decreasedupon treatment of the cells with all of the fucose analogs (100 μM).

For Lewis^(Y), of the cell lines examined, only untreated LS1745t andHL60cy had significant Lewis Y detected on the cell surface (cBR96staining) (FIG. 7B). The cBR96 detection of this structure wassignificantly decreased upon treatment of the cells with all of thefucose analogs (100 μM).

For P-selectin, of the cell lines examined, only untreated HL60cy hadsignificant P-selectin ligand detected on the cell surface. Thedetection of this ligand was decreased somewhat by treatment of thecells with all fucose analogs, except for alkynyl furocse peracetate(SGD-1890) (100 μM) (FIG. 7C). Untreated Ramos cells showed littleP-selectin ligand; however, upon treatment with the fucose analogs thesignal for this ligand increased. This is unusual and was not observedwith previous treatment of these cells with 2-fluorofucose (SGD-2083) oralkylnyl fucose (SGD-1887).

The results suggest that treatment with these fucose analogs can affectthe presence of fucose on the cell surface in general and alsospecifically the fucosylation of Lewis X and Lewis Y modifications onthe cell surface and sialyl LewisX present on the P-selectin ligand.

Example 6 Leukocytosis and Decreased E-selectin Binding Following OralDosing of 2-fluorofucose

The effects of a fucose analog on leukocytosis and E-selectin bindingwere eamined in mice. Female Balb/c mice were given oral 2-fluorofucose(SGD-2083) in the drinking water or left untreated. Mice were bled priorto dosing and then weekly for three weeks to assess circulating cellnumbers and their ability to bind E-selectin. In one. study,2-fluorofucose was formulated at 1 mM, 10 mM or 100 mM in the drinkingwater (n=3 per group). At day 14, mice were treated with TiterMAX®Classic adjuvant (Sigma) to stimulate polyclonal, antigen non-specificantibody production by B cells, and remained on the2-fluorofucose-containing water through day 21. In a second study, micewere given oral 2-fluorofucose formulated at 10 mM and 100 mM in thedrinking water for three weeks without any other treatments (n=6). Onday 21, a pool of lymph nodes (axillary, brachial, superficial inguinal,and mesenteric) from each of three animals was assessed in addition toblood. Lymph nodes were homogenized into single cell suspensions, andtotal cell numbers were determined by counting on a hemcytometer, usingTrypan Blue for dead cell exclusion. To determine total white cellnumbers/μL blood, samples of blood from individual animals were countedon a hemacytometer, using Turk's solution (0.01% gentian violet in 3%acetic acid) to exclude red blood cells (RBCs). RBCs were eliminatedfrom the remainder of the blood by osmotic lysis for flow cytometricanalysis. Cells were incubated with anti-Gr-1-FITC antibodies (BDBiosciences) to identify neutrophils, and a recombinant E-selectin-humanFc fusion protein (R&D Systems). Cells were washed and then incubatedwith a PE-labeled goat anti-human IgG-Fc secondary antibody (JacksonImmunoresearch) to detect bound E-selectin. Samples were collected on aFACSCalibur flow cytometer and analyzed using CellQuest software. Thepercentage of Gr-1+ cells was determined and absolute number ofneutrophils was calculated using the total white cell number from thehemacytometer count. In addition, flow samples were gated for Gr-1+cells to assess E-selectin binding to neutrophils by histogram analysis.The geometric mean of the E-selectin fluorescent signal was determinedfrom the histogram.

Results

The results in FIGS. 5A and 5B show that oral administration of2-fluorofucose (SGD-2083) resulted in an increase in circulating whiteblood cells and neutrophils, in a dose-dependent manner. 2-fluorofucosegiven at 1 mM had very little effect, whereas increasing effect wasobserved with increasing doses of 10 mM and 100 mM 2-fluorofucose. Thedata shown in FIGS. 5A and 5B are from the first study, day 14. Similarresults were obtained at days 7 and 21 in the first study as well asdays 7, 14, and 21 in the second study (data not shown). Lymph nodeswere also assessed at day 21 in the second study and FIG. 5C shows thatoral administration of 2-fluorofucose results in a marked decreased incellularity in the lymph nodes. The effect was more severe at 100 mMcompared to 10 mM.

Oral administration of 2-fluorofucose also results in decreased inE-selectin binding to neutrophils (FIG. 6). The effects of the fucosesanalogs was also dose-dependent, with 1 mM having little effect and 10mM and 100 mM having increasing effects (FIGS. 5B and 5C).

The observed increases in circulating white blood cells and neutrophils(leukocytosis) is consistent with the inhibition of E-selectin bindingby neutrophils. E-selectin mediates extravization of white blood cellsinto the periphery and lymph nodes, and inhibition of E-selectin binding(by inhibiting fucosylation) would also reduce extravization and resultin accumulation of white blood cells in the blood. These results suggestthat fucose analogs that inhibit protein fucosylation, and E-selecinfucosylation in particular, can act to inhibit autoimmunity.

Example 7 Tumor Growth Inhibition by Administration of Fucose AnalogsStudy 1

Human-derived cell lines were evaluated for their susceptibility to thefucose analog 2-fluorofucose in vitro. The cells lines were: LS174Tcolon adenocarcinoma, PC-3 colon adenocarcinoma, HL-60 acute mylogenousleukemia, Ramos Burkitt lymphoma, and Caki-1 renal cell carcinoma. Thecell lines were cultured in the presence of 100 μM 2-fluorofucose(SGD-2083) in growth media, 100 μM alkynylfucose (SGD-1887) in growthmedia, or control growth media (without a fucose analog) for two weeks.The growth media were MEM Eagle with 10% FBS (LS174T), 50:50 F12 andRPMI with 10% FBS (PC-3), RPM1 with 10% FBS (HL-60), IMDM with 10% FBS(Ramos), and McCoy with 10% FBS (PC-3). The cells were evaluated forcell surface fucosylation by FACS using antibody cBR96 to detect LewisY,antibody SSEA-1 to detect LewisX, P-selectin ligand to detectP-selectin, and AOL lectin to detect the general level of fucosylation.

Results:

The results of the FACS evaluation revealed variable levels offucosylated cell surface proteins on the different cell lines (data notshown). 2-fluorofucose (SGD-2083) was generally a better inhibitor ofprotein fucosylation than alkynyl fucose (SGD-1887).

Study 2

To further evaluate the activity of these fucose analogs, furtherstudies were performed in vivo using tumor cells that had beenpre-treated by culturing in the presence of a fucose analog, or usinguntreated tumor cells. Tumor cells were implanted into 10 mice per groupas follows. For the LS174-T, PC-3, and Caki-1 cell lines, 5×10⁵ cells in25% Matrigel were implanted subcutaneously into female nude mice. ForHL-60 and Ramos cell lines, 5×10⁶ cells were implanted subcutaneouslyinto female SCID mice. For mice implanted with untreated tumor cells,mice were provided regular drinking water. For mice implanted with tumorcells pre-treated with 2-fluorofucose (SGD-2083), the mice were provideddrinking water supplemented with 20 mM 2-fluorofucose (SGD-2083). Formice implanted with tumor cells pre-treated with alkylnyl fucose(SGD-1887), the mice were provided with regular drinking water. The micedid not drink water containing alkynyl fucose.

After 3 weeks of receiving 2-fluorofucose-containing drinking water,mice were returned to regular drinking water, except for mice withCaki-1 tumors. The latter mice were returned to regular drinking waterfor one week. After the week of receiving regular water, mice wererandomized to two groups of 5 each to receive drinking watersupplemented with 20 mM 2-fluorofucose or regular drinking water. Micewere sacrificed when tumors reached about 1000 mm³.

Referring to FIG. 8A-E, tumor growth inhibition in vivo was seen forLS174T, PC-3, and Caki-1 cells treated with 2-flurofucose (SGD-2083). Nochange in tumor growth was observed for HL-60 and Ramos cells. ForCaki-1, tumor growth inhibition was not observed during the firsttreatment period, but was observed after the mice were returned to2-fluorofucose treatment. For the other cell lines, tumor growthinhibition appeared to start when tumor size had reached about 150 mm³.The slower growing Caki-1 tumors did not reach this point until thesecond treatment period with 2-fluorofucose (SGD-2083). These resultsindicate that treatment with fucose analogs can inhibit tumor growth.

Study 3

In a third study, tumor cells were implanted without prior treatmentwith a fucose analog. LS174T colon adenocarcinoma cells (5×10⁵ cells in25% Matrigel) were implanted subcutaneously into female nude mice. Micewere supplied with 50 mM 2-fluorofucose (SGD-2083) in their drinkingwater from 7 days before implant until 21 days after implant, or weresupplied with regular drinking water.

Results

Referring to FIG. 8F, Mice given 50 mM 2-fluorofucose (SGD-2083) intheir drinking water showed a substantial inhibition of tumor growth,achieving an average tumor size of 110 mm³ versus 734 mm³ for micesupplied with regular drinking water. Collectively, these resultssuggest that administration of a fucose analog can inhibit tumor growth.

Example 8 Tumor Vaccine Model

Female Balb/c mice were immunized by subcutaneous implantation of 1million A20 murine lymphoma cells (killed by irradiation) on day −21 andday −7. Another group of mice were not given any immunization. On day 0,all mice were inoculated iv with 1.5 or 5 million live A20 cells. Ondays −14 through +21, mice were provided with 50 mM 2-fluorofucose(SGD-2083) in their drinking water or given regular drinking water. The8 treatment groups were as follows:

-   -   1. No immunization, 1.5 million live A20 cells, regular drinking        water    -   2. No immunization, 5 million live A20 cells, regular drinking        water    -   3. No immunization, 1.5 million live A20 cells, 50 mM SGD-2083        in drinking water    -   4.No immunization, 5 million live, A20 cells, 50 mM SGD-2083 in        drinking water    -   5. Immunized, 1.5 million live A20 cells, regular drinking water    -   6. Immunized, 5 million live A20 cells, regular drinking water    -   7. Immunized, 1.5 million live A20 cells, 50 mM SGD-2083 in        drinking water    -   8. Immunized, 5 million live A20 cells, 50 mM SGD-2083 in        drinking water

Results

Referring to FIG. 9A, the study design is shown. Referring to FIG. 9B,mice that did not receive any immunization succumbed to the live A20challenge from days 22-35. Mice receiving 2-fluorofucse (SGD-2083)survived a few days longer than those receiving regular drinking water.Two mice immunized with 5 million killed A20 cells and receiving regulardrinking water succumbed to the live A20 challenge. All mice receivingimmunization and 2-fluorofucose (SGD-2083) in their drinking water werestill alive at data collection.

The present invention is not limited in scope by the specificembodiments described herein. Various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims. Unless otherwise apparent from the context any step, element,embodiment, feature or aspect of the invention can be used incombination with any other. All patent filings, and scientificpublications, accession numbers and the like referred to in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if so individually denoted.

1-59. (canceled)
 60. A pharmaceutical composition comprising atherapeutically effective amount of a fucose analog selected from thegroup consisting of one of the following formulae (V) or (VI):

or a biologically acceptable salt or solvate thereof, wherein eachfucose analog of formula (V) or (VI) can be the alpha or beta anomer orthe corresponding aldose form; wherein each of R¹, R³, and R⁴ isindependently selected from the group consisting of —OH and —OC(O)C₁-C₁₀alkyl; and R² is F, R^(2a) and R^(3a) are each H, and R⁵ is —CH₃. 61.The pharmaceutical composition of claim 60, wherein each of R¹, R³, andR⁴ is independently selected from the group consisting of —OH and —OAc,R² is F, R^(2a) and R^(3a) are each H, and R⁵ is —CH₃.
 62. Thepharmaceutical composition of claim 60, wherein the fucose analog is2-deoxy-2-fluorofucose.
 63. The pharmaceutical composition of claim 60,wherein the fucose analog is 2-deoxy-2-fluorofucose peracetate.
 64. Thepharmaceutical composition of claim 60, wherein the composition issuitable for oral administration.